Epimedium acuminatum Franchet. by first conducting LC-MS analysis, leading to the identification of 32 compounds. Furthermore, guided by LC-MS profiling, three new 8-prenylated quercetin glycosides , one new anthocyanin (delphinidin-3-O-p-coumaroyl-sophoroside) and six known compounds were isolated from the flowers of E. acuminatum for the first time, and their structures were characterized based on spectroscopic methods including 1D and 2D NMR, and HRESIMS. Combining our discoveries and literature survey, a revised classification of Epimedium flavonols was proposed as Type A (8-prenylated kaempferol based), which was further subdivided into subtype icaritin and subtype demethylicaritin, and Type B (8-prenylated quercetin based), which was further subdivided into subtype 3′-hydroxylicaritin and subtype 3′-hydroxyldemethylicaritin. The structure-activity relationship (SAR) study was carried out by comparing testosterone production-promoting activities of all the new compounds along with nine related Epimedium flavonols, revealing that the new 8-prenylated quercetin glycosides (subtype 3′-hydroxyldemethylicaritin in Type B) exhibited lower testosterone production-promoting activities in rat primary Leydig cells than Epimedium flavonols of subtype demethylicaritin in Type A, but possessed higher activities than the Epimedium flavonols of subtype icaritin in Type A. These results suggested that either methylation at C-4′ position or hydroxylation at C-3′ position of ring B could significantly reduce the testosterone production-promoting activities of Epimedium flavonols.Phytochemical investigation was carried out for the flowers of   Epimedium L. is a medicinally important herbaceous genus in the family Berberidaceae containing more than 60 perennial plant species. The native occurrences of these plants are geographically discontinuous from North Africa (Algeria) to East Asia, with most of them endemic to China   . Among Eprovince . BesidesShennong Bencao Jing ” that Epimedium herbs were documented with main effects of treating sexual dysfunction, tonifying kidney Yang, bones and strength. Nowadays, modern pharmacological studies have verified a specific class of 8-prenylated flavonols in Epimedium plants as the major bioactive components (Epimedium flavonols), which exhibit extensive bioactivities of regulating hormone production, modulating immunological function, anti-rheumatic, anti-osteoporosis, anti-cancer, anti-aging, etc . However, the key difference is the presence or absence of hydroxyl group at C-3′ position of ring B. Depicted in Epimedium flavonols, among which Type A Epimedium flavonols with the backbone of 8-prenylated kaempferol are widely found in Epimedium species, including subtypes of icaritin (4′-methoxyl) and demethylicaritin (4′-hydroxyl)   . By contethoxyl)  and 3′-hethoxyl) , shown iEpimedium plants have been extensively examined and mainly found to produce Epimedium flavonols of icaritin subtype in Type A, and the rhizomes have been proven to principally accumulate Epimedium flavonols of demethylicaritin subtype in Type A  were thoroughly explored by combining LC-MS and NMR data. Guided by a preliminary LC-MS profiling, three new 8-prenylated quercetin glycosides (subtype 3′-hydroxyldemethylicaritin in type B), one new anthocyanin and six known flavonols were isolated from flowers of E. acuminatum, structures of which were determined through 1D and 2D NMR, and HRESIMS. Based on our discoveries and literature survey, a revised classification for Epimedium flavonols was proposed. Finally, the testosterone production-promoting activities of all the new compounds were tested and compared with nine related Epimedium flavonols, and their structure-activity relationship (SAR) were analyzed, revealing that Epimedium flavonols of subtype demethylicaritin in Type A possessed the highest potency for stimulating the testosterone production, and either methylation at C-4′ position or hydroxylation at C-3′ position of ring B could decrease the bioactivity. Combining structural characterization and SAR data, our findings could provide fundamental rules for understanding structural variations in Epimedium flavonols, potential candidates for medicinal use, and assist the selection of substrates for identification of prenyltransferases (PTs), UDP-glycosyltransferases (UGTs) and methyltransferases (MTs) in Epimedium plants.Until now, the leaves of n Type A . By cont flowers . Since fλmax at 520 nm for peaks 1, 3, 6, and 9 . Peak 2 was identified as petunidin-3,5-di-O-glucoside for showing molecular ions at m/z 641.1736 [M]+, and fragment ions at m/z 479.1200 [M-Glc]+ and m/z 317.1170 [M-Glc-Glc]+. Peak 5 was identified as petunidin-3-O-glucoside for giving molecular ions at m/z 479.1200 [M]+ and fragment ions at m/z 317.1170 [M-Glc]+.The ultraviolet-visible spectroscopy (UV-Vis spectra) showed the 6, and 9 , which w 2 and 5 , which wferences . Peak 1 λmax at around 240−280 nm and 350−360 nm for peaks 11−32 , peak 21 was identified as ikarisoside C for giving molecular ions at m/z 825.2831 [M+H]+, and fragment ions at m/z 663.2256 [M+H-Glc]+ and m/z 517.1721 [M+H-Glc-Rha]+; similarly, peak 22, 23, and 24 were identified as epimedoside E, diphylloside B, and epimedoside A with molecular ions at m/z 795.2740 [M+H]+, m/z 809.2872 [M+H]+, and m/z 663.2256 [M+H]+ , peak 25 was identified as epimedin A for producing molecular ions at m/z 839.3002 [M+H]+, m/z 677.2462 [M+H-Glc]+, m/z 531.1848 [M+H-Glc-Rha]+, and m/z 369.1346 [M+H-Rha-Rha-Glc]+; similarly, peak 26, 27 and 28 were identified as epimedin B, epimedin C and icariin, separately, and were confirmed with the authentic samples . Peaks 16−18, and 20 gave molecular ions at m/z 679.2239 [M+H-Glc]+ and fragment ions at m/z 533.1674 [M+H-Glc-Rha]+  was isolated as dark purplish red powder, the HRESIMS data  gave a molecular formula of C36H37O19+. The presence of major fragment ion at 303.0482 [M]+, in combination with the 1H NMR and 13C NMR data with characteristic proton chemical shifts shown in δH 4.80  and δH 5.34 , and both were detected to be d-glucose from acid hydrolysis experiments. The β-configuration of glucose moieties was assumed by the observed coupling constants (J = 7.6 and 7.5 Hz) (δH 5.34) of Glc A and H-1‴ (δH 4.80) of Glc B with C-3 (δC 145.0) and C-2″ (δC 84.1) of Glc A, respectively. Additionally, the aromatic signals of the 1,4-substituted benzene ring , and an ester group (δC 168.6), together with two olefinic protons signals exhibiting large coupling constants  indicated a p-coumaric acid in trans-configuration, which was further confirmed with HMBC correlations from H-2‴′ (δH 7.07) to C-7‴′ (δC 146.3), and from H-7‴′ (δH 7.09) to C-9‴′ (δC 168.6). The acylation of Glc B with p-coumaric acid was deduced from HMBC correlations of methylene protons  with C-9‴′ (δC 168.6). Therefore, the structure of 1 was determined as shown (Delphinidin 3- 7.5 Hz) . The linas shown , 4.2, peak 16) was obtained as yellow powder, HRESIMS data  gave a molecular formula of C38H48O21. The 1H NMR data , 1.71 , 5.18 , 3.42 , and 3.58 . The presence of three sugar moieties was deduced from anomeric hydrogen signals at 4.99 , δH 4.24 , and 5.50 , which was further confirmed to be d-glucose and l-rhamnose from acid hydrolysis. The large coupling constant observed with the anomeric hydrogens (J = 7.9 and 7.6 Hz) indicated a β-configuration for glucose moieties, whereas the α-configuration of rhamnose was deduced from the chemical shifts of C-3‴ (δC 70.2) and C-5‴ (δC 70.5) , 6.89 , and 7.41  in 2, rather than an AABB coupling system observed for ikarisoside C, revealing the presence of a hydroxyl group at C-3′ in 2. HMBC correlations of H-1‴ (δH 5.50) and H-1″ (δH 4.99) with C-3 (δC 134.5) and C-7 (δC 160.4), respectively, supported that the 8-prenylated quercetin was glycosylated with Rha at C-3 and Glc A at C-7. The Rha (2→1) Glc B linkage was confirmed with the HMBC correlation from H-1‴′ (δH 4.24) of Glc B to C-2‴ (δC 81.5) of Rha. Therefore, the structure of 2 was identified as shown (3′-hydroxylikarisoside C (NMR data  displayeC 70.5)   was isolated as yellow powder, HRESIMS data  gave a molecular formula of C37H46O20. The NMR data were similar to those of epimedoside E, except for an ABX coupling system at δH 7.32 , 6.89  and 7.41  in 3, instead of a AABB coupling system in epimedoside E, supporting the existence of a hydroxyl group at C-3′ in 3. The three sugar moieties were confirmed to be d-glucose, l-rhamnose and d-xylose from acid hydrolysis. The large coupling constant observed with the anomeric hydrogen (J = 6.7 Hz) indicated a β-configuration for the glucose moiety, whereas the α-configuration for rhamnose and β-configuration for xylose was deduced from chemical shifts of C-3‴ (δC 70.3) and C-5‴ (δC 70.4) for rhamnose, and C-3‴′ (δC 76.2) and C-5‴′ (δC 65.7) for xylose, respectively (δH 5.32) and H-1″ (δH 4.98) with C-3 (δC 134.2) and C-7 (δC 160.4), respectively, supported that the 8-prenylated quercetin was glycosylated with Rha at C-3 and Glc at C-7. The Rha (2→1) Xyl linkage was confirmed with the HMBC correlation from H-1‴′ (δH 4.16) of Xyl to C-2‴ (δC 80.1) of Rha. Therefore, the structure of 3 was elucidated as shown  was obtained as yellow powder, HRESIMS data  gave a molecular formula of C38H48O20. The NMR data were similar to those of diphylloside B, except for an ABX coupling system at δH 7.31 , 6.87  and 7.36  in 4, instead of a AABB coupling system in diphylloside B, supporting the existence of a hydroxyl group at C-3′ in 4. The presence of three sugar moieties was deduced from anomeric hydrogen signals at δH 4.99 , 4.88 , and 5.39 , which was further confirmed to be d-glucose and l-rhamnose from acid hydrolysis. The large coupling constant observed with the anomeric hydrogen (J = 7.7 Hz) indicated a β-configuration for glucose moiety, whereas the α-configuration of rhamnose moieties was deduced from the chemical shifts of C-3‴ (δC 70.3) and C-5‴ (δC 70.7) of Rha A, and C-3‴′ (δC 70.5) and C-5‴′ (δC 68.8) of Rha B (δH 5.39) and H-1″ (δH 4.99) with C-3 (δC 134.1) and C-7 (δC 160.5), respectively, supported that the 8-prenylated quercetin was glycosylated with Rha A at C-3 and Glc at C-7. The Rha A (2→1) Rha B linkage was confirmed from the HMBC correlation from H-1‴′ (δH 4.88) of Rha B to C-2‴ (δC 75.7) of Rha A. Therefore, the structure of 4 was identified as shown  (Epimedium flavonols was in the order: subtype demethylicaritin in Type A (1.29−1.56 fold) > subtype 3′-hydroxyldemethylicaritin in type B (1.15−1.31 fold), including the compounds 2−4 > subtype icaritin in Type A (1.12−1.18 fold). Significant difference (p = 0.015) was found between subtype demethylicaritin in Type A and subtype icaritin in Type A, but was not detected between subtype demethylicaritin in Type A and subtype 3′-hydroxyldemethylicaritin in Type B (p = 0.132), and between subtype 3′-hydroxyldemethylicaritin in Type B and subtype icaritin in Type A (p = 0.067), see  control , and theiability . At a do56 fold) . The tesE. acuminatum, a total of 32 compounds were identified, including 22 flavonols, six anthocyanins, three phenolic acids and one alkaloid. Among the 22 flavonols, 12 were found to be Type A Epimedium flavonols, four were identified as Type B Epimedium flavonols, and the remaining six were identified as non-prenylated flavonols. For Type A Epimedium flavonols, both of the subtypes (icaritin and demethylicaritin) were detected, whereas only flavonols of 3′-hydroxyldemethylicaritin subtype was observed for Type B. The petals and inner sepals of E. acuminatum were highly similar in chemical constituents, except that petunidin derivates and delphinidin-3,5-di-O-glucoside were only present in the inner sepals. The content of Epimedium flavonols in the petals was higher than that in the inner sepals. The LC-MS profiling led to the isolation of one new anthocyanin (1), three new 8-prenylated quercetin glycosides (Type B) (2−4), and six known flavonols (5−10) from the flowers of E. acuminatum.In our LC-MS analyses conducted separately on petals and inner sepals from flowers of 2−4), in combination with the previous discovered 3′-hydroxylepimedoside A  isolated in our study completed the Epimedium flavonols of subtype 3′-hydroxyldemethylicaritin in Type B, and Epimedium flavonols of subtype 3′-hydroxylicaritin in Type B still remains incomplete. Besides, for plant flavonoids, it is common for methylation to occur on the C-3′ position of ring B   . However species  from LC-Epimedium flavonols was measured and found to be different in petals, inner sepals, rhizomes, and leaves of E. acuminatum (Epimedium flavonols was higher than Type B. The relative content of Epimedium flavonols of subtype icaritin in Type A (4′-methoxyl) in different tissues was in the order: Leaves > rhizomes >> petals > inner sepals, whereas the highest content of subtype demethylicaritin in Type A (4′-hydroxyl) was found in rhizomes, followed by petals, inner sepals, and then leaves. Epimedium flavonols of subtype 3′-hydroxyldemethylicaritin in Type B (4′-hydroxyl) was highest in petals, and only trace amount was detected in inner sepals, rhizomes, and leaves, which should be reason why this subtype of Epimedium flavonols has remained elusive for long. On the whole, the different accumulation pattern of hydroxylated and methylated Epimedium flavonols in different tissues should indicate different biological functions in the corresponding tissues, which are worthy to be investigated. According to studies on biological functions of hydroxylated and methylated flavonoids in plants, hydroxylated flavonoids might contribute to the formation of colors and tastes of flowers which could facilitates pollination, whereas methylation improves chemical stability and membrane permeability, and maybe also the toxicity of flavonoids, which might help to resist microorganism and herbivores  and nine structurally related compounds were tested, which could be divided into three major types of Epimedium flavonols based on their chemical structures except for the new compound 1 , including icaritin and demethylicaritin subtypes, and Type B (8-prenylated quercetin based), including 3′-hydroxylicaritin and 3′-hydroxyldemethylicaritin subtypes. The SAR study was conducted by comparing testosterone production-promoting activities of new compounds with nine related Epimedium flavonols, suggesting that either methylation at C-4′ position or hydroxylation at C-3′ position of ring B could decrease the biological activity.Phytochemical investigation on petals and inner sepals from flowers of E. acuminatum were collected, and quick-frozen in liquid nitrogen in the spring of 2021. The samples were later freeze-dried and stored at −20°C until extraction. Voucher specimens were deposited at the Herbarium of the Institute of Medicinal Plant Development, Beijing, China (IMPLAD), and were identified by Professor Baolin Guo (IMPLAD).Plants were grown in the Germplasm Repository  at latitude 32.06 and longitude 131.31. Fresh petals, inner sepals, leaves, and rhizomes of 3OD and DMSO-d6. Semipreparative HPLC was conducted on a Lumtech K-501 equipped with a K-2501 UV detector. A 250 mm × 50 mm, 10 μm, ODS column and a 250 mm × 10 mm, 5 μm, ODS-A column  were applied for obtaining desired fractions and further purification, respectively. Macroporous adsorption resin D101 , and Sephadex LH-20 (40−70 μm)  were used for column chromatography. Chromatographic grade methanol and acetonitrile  were used for preparative HPLC. Analytical grade methanol and acetic acid  was used for extraction and column chromatography. Reference standards including delphinindin-3,5-di-O-glucoside, delphinidin-3-O-glucoside, petunidin-3-O-glucoside, neochlorogenic acid, chlorogenic acid, and astragalin were purchased from Sigma-Aldrich Co., Ltd. , and reference standards of hyperoside, isoquercitrin, astragalin, magnoline, epimedins A−C, icariin and 2″-O-rhamnosylicariside II were purchased from Chengdu PUSH-biotechnology Co., Ltd. . Other reference standards used in this study were isolated and their structures were confirmed through a comparison of NMR and MS data with the literature.High-resolution mass spectrometry was conducted on a Waters Acquity UPLC I-Class Plus-Xevo G2 XS Q-ToF , equipped with an ESI source. The separation was carried out with a RP-C18 column (150 mm × 3.0 mm) with particle size of 1.8 µm . The NMR spectra were measured on a Bruker Avance III 400 MHz and a 500 MHz spectrometer , referenced to the solvent signals of CDE. acuminatum, separately, were extracted with MeOH (containing 0.1% acetic acid) at 4°C and were centrifuged for 10 min at 10,000 rpm. The supernatants were collected, dried with vaccum centrifuge concentrator , and stored at −20°C until analysis. The dried extracts were re-dissolved in MeOH right before analysis and were examined on a liquid chromatography quadrupole time-of-flight mass spectrometer, Q-TOF-MS (Waters Xevo G2-XS QTOF)  with an ESI source consisting of ACQUITY UPLC I-Class instrument . The separation was carried out with a RP-C18 column  at 40°C. The elution program consisted 0.1% formic acid (A) and acetonitrile (B) as the mobile phases, and a gradient elution profile was applied (flow rate of 0.4 ml min−1): 0−1 min (5% B), 1−8 min (5%–30% B), 8−12 min (30%–40% B), 12−16 min (40%–95% B), 16−17 min (95%–100% B), 17−21 min (100% B), 21−22 min (100% B−5% B), 22−25 min (5% B). The mass spectrometer operated in positive ion mode. ESI source parameters were as follows: capillary voltages 0.5 kV; desolvation gas flow 1000 L Hr−1 (N2). MS spectra were obtained over the range of m/z 100−1,200.The samples of petals, inner sepals, leaves and rhizomes from Epimedium flavonols was calculated from peak areas of samples based on the intensity of the corresponding standard compounds.The compounds in sample extracts were identified by comparing with the retention times, characteristics of UV-Vis spectra of peaks and the mass spectrometric information of reference standards using software MassLynx v4.1. The relative content of 12 key E. acuminatum (100 g) were extracted in ultrasonic bath (1 h) for three times with 80% MeOH (containing 1% acetic acid) at room temperature in darkness. The mixture was filtered through a Buchner funnel and the pooled filtrates were concentrated under reduced pressure at 45°C. The extract was then partitioned against petroleum ether, and concentrated to obtain the crude anthocyanin-flavonol containing MeOH-aqueous extract.A mixture of dried petals and inner sepals of 3CN (A) and 0.5% acetic acid (B). The elution profile consisted of 15% A for 25 min, and 20% A for 30 min to obtain three desired fractions (Fr.1−3).The crude extract (82.0 g) was firstly passed through a macroporous adsorption resin D101 (un-polarity) with distilled water to remove most of the polysaccharides, and then the solvent was changed to 50% EtOH to elute anthocyanins and desired flavonols. The 50% EtOH residue (5.93 g) was first separated by semipreparative HPLC with a 250 mm × 50 mm, 10 μm, ODS column using solvents CH1 (105.0 mg).Fr.1 (182.5 mg) was further separated by passing through Sephadex LH-20 (eluted with 40% MeOH) to yield compound 3CN (A) and 0.025% formic acid (B). The elution profile consisted of a linear gradient from 17% to 21% A in 20 min, isocratic elution for the next 30 min, followed by 100% A for 15 min at a flow rate of 2.0 ml/min to afford compound 2 , compound 3  and compound 4 .Fr.2 (176.6 mg) was first separated by passing through Sephadex LH-20 with MeOH to obtain three subfractions (Fr.2A−2C). Fr.2A (53.1 mg) was further purified by semipreparative HPLC with a 250 mm × 10 mm, 5 μm, ODS-A column  using solvents CH5  and 6 , respectively. 80.9 mg of Fr.3 (243.2 mg) was subjected to semipreparative HPLC with a 250 mm × 10 mm, 5 μm, ODS-A column  using solvents  at 2.0 ml/min to provide compound 7 , 8 , 9 , and 10 .Fr.2B (61.3 mg) and 20.5 mg of Fr.2C (50.8 mg) was separated using the same elution profile as Fr.2A, yielding compound O-p-coumaroyl-sophoroside (1): dark purplish red powder. HRESIMS m/z 773.1924 [M]+ . 1H and 13C NMR (CD3OD-DCl) data, see Delphinidin-3-(2): yellow powder. HRESIMS m/z 841.2773 [M+H]+ . 1H and 13C NMR (DMSO-d6) data, see 3′-hydroxylikarisoside C (3): yellow powder. HRESIMS m/z 811.2742 [M+H]+ . 1H and 13C NMR (DMSO-d6) data, see 3′-hydroxylepimedoside E (4): yellow powder. HRESIMS m/z 825.2897 [M+H]+ . 1H and 13C NMR (DMSO-d6) data, see 3′-hydroxyldiphylloside B 5) (6) (7) (8) (9) (10) (Six known flavonols were characterized as 3′-hydroxyl-epimedoside A (5) , hyperos5) (6) , ikariso (6) (7) , epimedo (7) (8) , diphyll (8) (9) , and epi(9) (10)  through 1–4 (each 1.0 mg) were dissolved in 6 mol L−1 CF3COOH (1 ml) and heated at 90°C for 2 h and cooled to room temperature. The hydrolysate was extracted with CHCl3 for three times, and the aqueous layer was concentrated to obtain the residue containing sugars. The obtained residues were dissolved in pyridine (200 μl), and L-cysteine methyl ester hydrochloride (1 mg) was added and heated at 60°C for 1 h. Subsequently, o-tolyl isothiocyanate (10 μl) was added and heated at 60°C for another 1 h  from 20% to 30% for 8 min (flow rate: 0.6 ml/min). The atomization temperature and wave filtering time were set at 35°C and 1 s, respectively. For compound 2 and 4, derivatives of l-rhamnose and d-glucose were detected. However, for compound 3, d-xylose was also observed. For compound 1, only derivative of d-glucose was detected .Standard sugars and compounds ther 1 h . After t6/ml with 2 ml culture medium in 6-well plates at the conditions: 37°C with 5% CO2. During the period of cell culture, the morphological changes of cells were monitored with an inverted microscope . To measure the purity of rat primary Leydig cells, 3β-HSD staining method was used , and the negative control samples were set without any compounds. For each experimental group, three replicates were set. During the assay, the diluted MTT solution was added to each well and incubated for 4 h. The supernatant was discarded, 100 μl of DMSO was added to each well, and the absorbance was measured at 570 nm by a microplate reader . Testosterone secreted into the culture medium was measured using testosterone ELISA kits according to the manufacturer’s instructions .The cellular viability was evaluated using the MTT proliferation assay. In the MTT proliferation assay, the positive control samples were set with the treatment of forskolin (5 μmol Lvia a two-tailed student’s t-test with two-sample equal variance.Statistical analysis of the measurement of testosterone was performed


Size exclusion chromatography and infrared spectroscopy were also used. Interestingly, HSQC analysis showed substantial changes in the oxygenated aliphatic region of lignins, showing an almost complete absence of signals corresponding to side-chains due to laccase depolymerization. Simultaneously, a significant loss of aromatic signals was observed by HSQC and 1H NMR, which was attributed to a deprotonation of the lignin benzenic rings due to polymerization/condensation by laccase reactions. Then, condensed structures, such as α-5′, 5-5′, and 4-O-5′, were detected by HSQC and 13C NMR, supporting the increment in molecular weight, as well as the phenolic content reduction determined in lignins.The usage of laccases is a sustainable and environmentally friendly approach to modifying the Kraft lignin structure for use in certain applications. However, the inherent structure of Kraft lignin, as well as that resulting from laccase modification, still presents challenges for fundamental comprehension and successful lignin valorization. In this study, bacterial and fungal laccases were employed to modify eucalypt Kraft lignin. To evaluate the type and range of the chemical and structural changes of laccase-treated lignins, different NMR techniques, including solution  Lignin is the second most abundant biological macromolecule on earth, giving lignocellulose biomass rigidity, water impermeability, and protection against microbial decay and mechanical stress . CurrentThe valorization of Kraft lignins largely depends on tailoring their physico-chemical properties for the different applications intended, including molecular weight distribution and presence of reactive groups. Different chemical treatments, such as oxidative modifications, can be used to change these Kraft lignin properties . However1H NMR and 2D NMR (based on heteronuclear single quantum correlation (HSQC)), and solid-state CP/MAS (cross polarization/magic angle spinning) 13C NMR, were used to investigate the type and range of the chemical and structural changes of eucalypt Kraft lignin, produced by two different laccases of bacterial and fungal origin. In addition, size exclusion chromatography (SEC) and Fourier transform infrared (FTIR) spectroscopy were also employed.The inherent structure of Kraft lignin, as well as that resulting from laccase polymerization, still present challenges for fundamental comprehension and successful valorization of lignin. Then, some of the most powerful tools for the structural characterization of lignin, such as multidimensional nuclear magnetic resonance (NMR) spectroscopy, are being applied for this purpose ,8,14,18.Streptomyces ipomoeae (SiLA) and Myceliophtora thermophila (MtL), respectively, were used to polymerize Kraft lignin from Eucalyptus globulus. These enzymes show a variety of properties, such as stability at alkaline pH and high temperature, that make them appropriate for lignin polymerization, as it has been previously shown [1H NMR, 2D NMR (HSQC), and CP/MAS 13C NMR, were used to investigate the Kraft lignin, as well as the type and range of the chemical and structural changes produced by laccase enzymes.In this study, bacterial and fungal laccases, namely, from ly shown ,16. SubsAs expected, similar to trends in previous studies ,22, a hi®, 1 IU/mg lignin for 6 h) to oxidize different lignin fractions derived from birch and spruce alkali lignins, observing an important total phenolic content reduction (between 30% and 70%). Mayr et al. [Trametes villosa and Trametes hirsuta (90 IU/g lignin for 83 h), respectively. Finally, MtL laccase (1333 IU/g lignin for 2 h) produced a phenolic content reduction of eucalypt Kraft lignin around 66% [The phenolic content reduction of different lignin side-streams has been described either by bacterial or fungal laccases. Wang et al.  reportedr et al.  also shor et al.  reportedIncreasing the laccase dosage to 100 IU/g of lignin and the incubation time to 240 min resulted in a mild further phenolic content decrease of the resulting laccase-treated lignins. Then, SiLA produced a phenolic content decrease of 65.7% (SiLA-KL2), whereas a phenolic content reduction of 38.6% was achieved by MtL laccase (MtL-KL2). In a previous work by Mayr et al. , a higheWeight-average (Mw) and number-average (Mn) molecular weights, as well as polydispersity (Mw/Mn) values, showed in ®). Mayr et al. [T. versicolor (0.75 IU/mg lignin for 24 h). Huber et al. [The molecular weight increment of different lignin side-streams has been described either by bacterial or fungal laccases. Wang et al.  describer et al.  reportedr et al.  showed ir et al.  achievedr et al.  showed aAn increase in laccase dosage (100 IU/g of lignin) and reaction time (240 min) also led to a mild further increase in the Mw values. Then, SiLA laccase produced a 2.4-fold increase of the molecular weight (SiLA-KL2), whereas a 2.2-fold increase was observed when MtL laccase was used (MtL-KL2). Previously, Mayr et al.  also ach−1 (aromatic ring breathing in S and G condensed units), 1270 cm−1 (aromatic ring breathing with C=O stretching in G units), 1220 cm−1 , 1115 cm−1 (C–H bond deformation in S units), 1025 cm−1 (C–H bond deformation in G units), and 820 cm−1 (C–H out of plane deformation in S units) were observed. Moreover, the characteristic bands associated to the lignin aromatic skeleton  and to the C−H asymmetric vibrations and deformation (1455 cm−1) were also seen.−1  were tentatively endorsed to cellulose and hemicelluloses. On the other hand, a shoulder at 1715 cm−1 was attributed to the carbonyl groups of hemicelluloses. Nevertheless, this band can be also associated to the unconjugated C=O stretching resulting from lignin oxidation [According to the composition of untreated lignin , severalxidation . −1 (C=O stretching for unconjugated linkages) and at 1650 cm−1 (C=O stretching for conjugated linkages)  –e, especinkages) . This obh et al.  also desh et al.  of diffei et al.  attributOn the other hand, laccase-treated lignins did not show modifications in the bands associated to the lignin aromatic skeleton, in the same way as that observed by Gouveia et at.  when MtLC/δH 0.0–150.0/0.0–9.0) (C/δH 45.0–95.0/2.5–6.0 ppm) spectrum (C/δH 90.0–150.0/5.0–9.0 ppm) spectrum  a, the oxspectrum b, and thspectrum c. The maC/δH 0.0–50.0/0.0–5.0 ppm) displayed a variety of signals. Some of them could be associated to extractives, while others could be attributed to groups neighboring alkene and oxygen-containing groups, such as ethers, carbonyl, and alcohol, which could originate from lignin degradation [The non-oxygenated aliphatic region (around δradation .α–Hα (Bα), Cβ–Hβ (Bβ), and the double (Bγ)) (3.5 linkages per 100 aromatic units). Other signals attributed to β-O-4′ (Cα–Hα for β-O-4′ S lignin units (Aα) and Cγ–Hγ (Aγ)) were also observed, although in much lower abundance (0.80 linkages per 100 aromatic units). The native resinol substructures, with C−C linkages, are usually stable to the alkaline conditions of Kraft pulping [α–Hα (Eα) and Cα′–Hα′ (Eα′)) (1.2 linkages per 100 aromatic units) and cinnamyl alcohol end-groups (Cγ–Hγ (Iγ)) (0.8 linkages per 100 aromatic units), were also recognized. On the other hand, Kraft-derived substructures could also be found in this region. Among them, aryl-glycerol substructure, resulting from the alkaline breakdown of non-phenolic β-aryl ether linkage during Kraft pulping [α–Hα (AGα), Cβ–Hβ (AGβ), and Cγ–Hγ (AGγ)) (2.1 linkages per 100 aromatic units). Overlapping with the Cα–Hα correlation signal of aryl-glycerol, a Cα–Hα correlation signal of lignin terminal structures with a carboxyl group in Cβ , could also be identified (2.1 linkages per 100 aromatic units). Correlation signals from epiresinols (Cα–Hα (B′α) (overlapping with Cα–Hα (Eα) from spirodienones), Cβ–Hβ (B′β), and Cγ–Hγ (B′γ)) and diaresinol (Cβ–Hβ (B′′β)) were also found (0.8 linkages per 100 aromatic units). These diastereomers are derived from the transformation of native resinol substructure during Kraft pulping [The oxygenated aliphatic region of the untreated Kraft lignin spectrum was highly enriched in signals from β-β′ resinol substructures , C3–H3 (X3), C4–H4 (X4), and C5–H5 (X5)  b, togeth–H5 (X5) a.2,6–H2,6 ), G (C2–H2 (G2), C5–H5 (G5), and C6–H6 (G6)), and H lignin units ) ) from syringaldehyde or acetosyringone, and oxidized G units (C2–H2 (G′2) and C6–H6 (G′6) and C6–H6 (G′′6)) attributed to vanillin and acetovanillone, could also be observed. On the other hand, correlation signals corresponding to Kraft-derived lignin linkages were also found in the aromatic region. They included signals assigned to β1 stilbene (Cα–Hα (SB1α)) (2.2 linkages per 100 aromatic units) and β5 stilbene (Cβ–Hβ (SB5β) (0.1 linkages per 100 aromatic units), both resulting from the degradation of spirodienone and β-5′ phenylcoumaran during the Kraft pulping, respectively [2,6 in S1-1′ , G2 and G6 in G1-1′ (3-dimethoxy-para-diphenol), and S2,6 in S1-G1′/G5′ were also tentatively assigned as a result of Cα-C1 cleavage in a retro-aldol reaction, followed by a radical coupling reaction during Kraft pulping [In the same way as FTIR analysis , the aro ) c. The S/ectively ,40. Sign pulping ,38,39.γ) and diaresinol (B″β) were still observed, whereas signals from carbohydrates remained practically unaltered by both laccases. Similar observations were displayed for both laccases when the enzyme dosage and reaction time were increased to 100 IU/g and 240 min, respectively (®) bacterial laccase. Sun et al. [Trametes villosa and Trametes hirsuta laccases. n et al.  also desn et al.  also desα) condensed structures, which could result from lignin condensation/polymerization reactions by laccases action. In this sense, Wang et al. [® laccase, proposing different mechanisms for the formation of this condensed structure. On the one hand, the carbocation at Cα resulting from the cleavage of side-chains by laccases, mainly from β-β′ resinol substructures, could condense with the electron-rich G5 position, among others [α, could react with the electron-rich G5 position, among others [Simultaneously with the disappearance of side-chain signals, a new signal could be found in the aliphatic oxygenated region of the SiLA-KL1 spectrum, at 40 IU/g and 90 min b, and ofg et al.  already g others . On the g others ,31.13C–1H correlation signals was observed for laccase-treated lignins  showed strong bands at δC 147 ppm, attributed to C3 and C5 in S units (phenolic) and C3 and C5 in G units, and at δC 133 ppm, associated to C1 and C4 in phenolic S units and C1 in phenolic G units. This high content of phenolic units in eucalypt Kraft lignin observed by 13C NMR supported the high phenolic content determined by the Folin–Ciocalteu reagent , particularly in the SiLA lignin samples  in 4-O-5′ structures resulting during laccase (T. villosa) treatment of lignosulfonates, whereas Magina et al. [C 128 ppm to the C5 in 5-5′ structure formed during MtL laccase treatment of lignosulfonates. Therefore, these observations showed by 13C NMR also confirm the deprotonation of the lignin benzenic rings seen by HSQC and 1H NMR as a consequence of polymerization/condensation reactions by laccase enzymes, supporting the increment of molecular weight values  lignin was isolated by acid precipitation (pH 2.5) from Kraft black liquor, kindly supplied by La Montañanesa pulp mill . The chemical composition, determined by the Laboratory Analytical Procedures (LAP) [es (LAP) , was as Streptomyces ipomoeae CECT 3341.16 [Myceliophtora thermophila (Novozym® 51003), the latter kindly supplied by Novozymes . Enzyme activities  were estimated by oxidation of 5 mM 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) to its cation radical (ε436 = 29 300 M−1 cm−1) in 0.1 mM sodium acetate (pH 5) at 24 °C.Two different laccases from bacterial and fungal origin were used: a recombinant bacterial laccase (SiLA) from  3341.16  and a coAll the reagents used were of analytical grade, purchased from either Sigma–Aldrich  or Merck .A solution of 1.5 g/L of Kraft lignin was prepared in phosphate buffer at pH 7.0 and 8.0 (100 mM), according to optimal activity values for MtL and SiLA laccases, respectively ,49. Two 2CO3, was measured using a UV–Vis spectrophotometer . A calibration curve prepared from a standard solution of gallic acid (1–200 mg L−1) was used to quantify the total phenolic content of lignin samples, which was expressed as mg gallic acid equivalent (GAE) g−1 of lignin (on a dry basis).The total phenolic content of lignin samples was determined according to Jiménez-López et al. . Firstly−1 in NaOH (0.05 M). Two columns  coupled in series  and a safeguard column (35 × 7.8 mm) were used. The operational conditions were described elsewhere [SEC analysis (weight-average (Mw), number-average (Mn) molecular weights, and polydispersity (Mw/Mn)) were carried out in an Agilent Technologies 1260 HPLC ) at 254 nm. Previously, lignin samples were dissolved at a final concentration of 0.5 g Llsewhere .−1, 400 scans, and a spectral range of 2000–600 cm−1 [FTIR spectra of lignin samples were obtained by a JASCO FT/IR 460 Plus spectrometer , equipped with an accessory single reflection diamond, working with a resolution of 1 cm600 cm−1 .13C nuclear magnetic resonance (13C NMR) analyses of lignin samples were carried out in a Bruker Avance III 400MHz  at 100.64 MHz with the cross polarization/magic angle spinning (CP/MAS) technique. The operational conditions were described elsewhere [1H NMR and heteronuclear single quantum correlation (HSQC) (2D NMR), were recorded on 40 mg of lignins (dissolved in 0.75 mL of deuterated dimethylsulfoxide (DMSO-d6)) in a Bruker AVANCE 500 MHz , according to Eugenio et al. [C/δH 39.6/2.5 ppm). Abundance of β-O-4′, resinols, spirodienones, arylglicerol, epiresinols, diaresinols, and Ar−CHOH−COOH substructures was estimated by 2D-NMR from Cα–Hα correlations. Cinnamyl alcohol end-groups using Cγ–Hγ correlations, stilbenes (SB1 and SB5) using Cα–Hα correlations, C2,6–H2,6 correlations from S units, and C2–H2 correlations from G units were used to estimate the S/G lignin ratios.Solid-state lsewhere . Solutioo et al. . Residua1H NMR after treatment with both laccases, which was attributed to polymerization by laccase reactions. Then, condensed structures, such as α-5′, 5-5′, and 4-O-5′, could be observed by HSQC and 13C NMR, supporting the increment in molecular weight and polydispersity values, as well as the reduction in the phenolic content, determined in lignins treated by both laccases.Laccases oxidize phenolic lignin, leading to inter-unit linkages cleavage and, consequently, depolymerization. Simultaneously, the phenoxy radicals formed by the action of the laccase enzymes on the phenolic units, together with those derived from the cleavage of inter-unit linkages, undergo radical–radical coupling through phenyl ether-carbon and carbon-carbon links, resulting in new condensed structures. NMR characterization of eucalypt Kraft lignin treated by bacterial and fungal laccases provided structural changes that support both degradation and polymerization reactions. Compared to untreated lignin, HSQC analysis showed an almost complete absence of signals corresponding to lignin side-chains by laccase depolymerization. Simultaneously, a substantial diminution of aromatic signals was noticed by HSQC and


The binding of proteins to Z-DNA is hard to analyze, especially for short non-modified DNA, because it is easily transferred to B-DNA. Here, by the hybridization of a larger circular single-stranded DNA (ssDNA) with a smaller one, an LR-chimera (involving a left-handed part and a right-handed one) with an ssDNA loop is produced. The circular ssDNAs are prepared by the hybridization of two ssDNA fragments to form two nicks, followed by nick sealing with T4 DNA ligase. No splint (a scaffold DNA for circularizing ssDNA) is required, and no polymeric byproducts are produced. The ssDNA loop on the LR-chimera can be used to attach it with other molecules by hybridization with another ssDNA. The gel shift binding assay with Z-DNA specific binding antibody (Z22) or Z-DNA binding protein 1 (ZBP1) shows that stable Z-DNA can form under physiological ionic conditions even when the extra ssDNA part is present. Concretely, a 5′-terminal biotin-modified DNA oligonucleotide complementary to the ssDNA loop on the LR-chimera is used to attach it on the surface of a biosensor inlaid with streptavidin molecules, and the binding constant of ZBP1 with Z-DNA is analyzed by BLI (bio-layer interferometry). This approach is convenient for quantitatively analyzing the binding dynamics of Z-DNA with other molecules. Usually, research on Z-DNA, one of the basic and very interesting secondary DNA structures with left-handed helix and the zig-zag phosphate skeleton, is elusive and controversial ,2. Much 11- and d(CG)16-inserted plasmids to study the binding site and their affinity to the Z-DNA-specific binding antibody (Z22) by foot-printing using diethylpyrocarbonate .,34.n seqTo obtain cc-91B A, we hyb2 was present in the gel, indicating again that Z-DNA can form at relatively low ionic strength. Accordingly, we have successfully prepared LR-chimeras with an ssDNA hook involving either an APP-rich sequence or an APP-poor sequence for the Z-DNA part. The LR-chimeras are stable under low ionic strength . The ssDNA hook can be used to hybridize to another ssDNA strand to attach the LR-chimera to a surface for further analysis.Z22 and ZBP1 proteins were also used to verify the presence of the Z-DNA duplex in cc-91B formed mainly by non-APP sequences D−F. With2, 0.1% BSA, and 0.02% Tween-20 (BSA and Tween-20 avoid non-specific binding). The real-time binding curves of various concentrations of ZBP1 and LR-chimera are show in A linear ssDNA attaching 5’-terminal biotin was used to immobilize the LR-chimera to the biosensor surface coating with streptavidin A. The hyon, dissociation rate Koff, and affinity constant KD (Koff/Kon) were determined to be 8.28 × 103 M−1s−1, 4.68 × 10−6 s−1, and 0.566 nM, respectively. The R2 for fitting was as high as 0.9996. Accordingly, the binding of Z-DNA-specific proteins to Z-DNA could be investigated by using the prepared Bait at various conditions.2. This is also consistent with the electrophoresis result of ZBP1 binding to cc-91B (The binding of ZBP1 to cc-91B (APP-poor sequence) was also investigated D. Even wo cc-91B E. We can2) is usually necessary for the stabilization of non-modified linear Z-DNA in vitro, at which ZBP1 or other proteins are easily denatured. For our approach, the circular LR-chimera can form stable Z-DNA at low salt concentration, which is an important prerequisite for a ZBP1 binding assay. Therefore, for the first time, the binding curves of unmodified Z-DNA with both APP-rich and APP-poor sequences to ZBP1 were obtained in the presence of 10 mM MgCl2. This strategy makes it easy to study the binding dynamics of Z-DNA of unmodified arbitrary sequences with various proteins or other molecules, and the investigation of ionic effects becomes much more convenient.As we know, an extremely high salt concentration . The binding constant of ZBP1 to Z-DNA was also obtained (0.566 nM) in the presence of 10 MgClT4 DNA ligase, exonuclease I, and exonuclease III were purchased from Thermo Scientific . The Ultra GelRed (a dye staining both dsDNA and ssDNA) was purchased from Vazyme . The Z-DNA-specific antibody (Z22) was from Absolute Antibody Ltd. , and ZBP1 (recombinant Z-DNA-binding protein) was from Wuhan USCN Business Co., Ltd. . The streptavidin (SA) biosensor was purchased from SAIDELISI . All other chemicals were from Sigma-Aldrich .All oligonucleotides used in this study  were added into 7.0 µL H2O. After being heated to 90 °C for 3 min, the solution was cooled gradually (0.1 °C/s) to 37 °C and kept for 20 min. Then, 1.0 µL T4 DNA ligase (5 U) was added. The ligation reaction  was carried out at 37 °C for 2 h. The ligase was deactivated by incubation at 65 °C for 10 min. The preparation of c132A was the same as above. For the preparation of c92B, 1 μL of linear ssDNA ln-92a (40 μM) and ln-92b (40 μM), 2 µL of 1 × T4 DNA ligase buffer , and 3 µL of Spn92a (40 μM) and Spn92b (40 μM) were added into 9 µL H2O. After being heated to 90 °C for 3 min, the solution was cooled gradually (0.1 °C/s) to 37 °C and kept for 20 min. Then, 1 µL T4 DNA ligase (5 U) was added. The ligation reaction  was carried out at 37 °C for 2 h. The preparation of c132B used 1 × T4 DNA ligase buffer , and other operations were the same as for c92B.For the preparation of c92A, 5.0 μL of linear ssDNA l-92a (40 μM) and l-92b (40 μM), and 2.0 µL of 0.5 × T4 DNA ligase buffer , 1.0 µL of 10 × exonuclease III reaction buffer , 1.0 µL of exonuclease I (20 U), and 0.5 µL of exonuclease III (20 U) were added, then the digestion reaction was carried out at 37 °C for 12 h. Exonuclease I and III were deactivated at 80 °C for 15 min. The proteins and ions in the above solution were removed with phenol/chloroform and ethanol. Finally, the obtained circular DNA samples were stored in the buffer of 0.1 × TE  and analyzed using a Nanodrop 2000 spectrophotometer .To the above reaction containing the circular ssDNA solution (20 µL), 1.0 µL of 10 × exonuclease I reaction buffer  were added into 9.6 μL H2O. After being heated to 90 °C for 3 min, the solution was cooled gradually (0.1 °C/s) to 50 °C and kept for 2 h, and then continued to cool to 25 °C and was kept for 20 min. Finally, 2.4 µL of linear ssDNA L’40 (10 μM) was added to the above solution and incubated at 25 °C for 12 h . For cc-91B, 2.0 μL of c92B (10 μM), 2.0 μL of c132B (10 μM), and 2 μL of 10 × PBS buffer (containing MgCl2) were added into 12.0 μL H2O. After being heated to 90 °C for 3 min, the solution was cooled gradually (0.1 °C/s) to 25 °C and kept for 20 min . LR-chimeras with an ssDNA part were analyzed by 8% PAGE at 20 °C.For cc-91A, 2.0 μL of c92A (10 μM), 2.0 μL of c132A (10 μM), 2.0 μL of L59 (10 μM), and 2 μL of 10 × PBS buffer containing MgClB50 (5 μM), 2.0 μL of 10 × PBS buffer (containing MgCl2), and 6.0 μL of H2O were mixed. The solution was heated at 90 °C for 3 min, gradually cooled (0.1 °C/s) to 50 °C and kept for 2 h, and then continued to cool to 25 °C and was kept for 20 min. The product was assigned as Bait-cc-91 (containing left-handed DNA). For Bait-cl containing only B-DNA, 4.0 μL of l92 (5 μM), 4.0 μL of c132 (5 μM), 4.0 μL of LB50 (5 μM), 2.0 μL of 10 × PBS buffer (containing MgCl2), and 6.0 μL of H2O were mixed. The solution was heated at 90 °C for 3 min and gradually cooled (0.1 °C/s) to 25 °C and kept for 20 min. All products were analyzed by 6% PAGE at 20 °C.For a 20 μL reaction system, 4.0 μL of c92 (5 μM), 4.0 μL of c132 (5 μM), 4.0 μL of L2) (pH 7.2), 0.1% BSA, and 0.02% Tween-20. The response data and affinity parameter (KD) were obtained using the Octet Data Analysis Software Version 11.1.The affinity of ZBP1 protein binding to the non-modified Z-DNA was evaluated using BLI and an Octet Red 96 system  at 25 °C. The assay procedure included five steps: baseline (60 s); loading (180 s); baseline 2 (180 s); association (480 s); and dissociation (480 s). Z-DNA was 20 nM; ZBP1 were 25, 50, 100, 200, and 400 nM. The buffer used contained 1 × PBS buffer (containing 10 mM MgCl


However, little is known about the molecular events that regulate the onset of this important tumour-suppressive barrier. Here we identified the innate immune sensor Z-DNA binding protein 1 (ZBP1) as a regulator of the crisis program. A crisis-associated isoform of ZBP1 is induced by the cGAS–STING DNA-sensing pathway, but reaches full activation only when associated with telomeric-repeat-containing RNA (TERRA) transcripts that are synthesized from dysfunctional telomeres. TERRA-bound ZBP1 oligomerizes into filaments on the outer mitochondrial membrane of a subset of mitochondria, where it activates the innate immune adapter protein mitochondrial antiviral-signalling protein (MAVS). We propose that these oligomerization properties of ZBP1 serve as a signal amplification mechanism, where few TERRA–ZBP1 interactions are sufficient to launch a detrimental MAVS-dependent interferon response. Our study reveals a mechanism for telomere-mediated tumour suppression, whereby dysfunctional telomeres activate innate immune responses through mitochondrial TERRA–ZBP1 complexes to eliminate cells destined for neoplastic transformation.Cancers arise through the accumulation of genetic and epigenetic alterations that enable cells to evade telomere-based proliferative barriers and achieve immortality. One such barrier is replicative crisis—an autophagy-dependent program that eliminates checkpoint-deficient cells with unstable telomeres and other cancer-relevant chromosomal aberrations Dysfunctional telomeres activate innate immune responses through mitochondrial TERRA–ZBP1 complexes to eliminate cells that are destined for neoplastic transformation. Senescence is p53 and RB dependent and occurs when shortened telomeres elicit a DNA-damage response5. Loss of cell cycle checkpoints renders cells incapable of senescence activation, resulting in continued proliferation and telomere shortening. Such cells eventually succumb to crisis, characterized by extensive cell death and genome instability7. Crisis provides a redundant tumour-suppressor mechanism for replicative senescence, whereby CGAS–STING signalling triggers a non-canonical form of autophagy capable of executing cell death rather than sustaining cell survival1. Although this discovery established an essential role for autophagy during cell death in crisis, it remained unclear how dysfunctional telomeres engage nucleic-acid-sensing machineries and activate innate immune signalling pathways that are required for cell death.Replicative senescence and crisis constitute two anti-proliferative barriers that human cells must evade to gain immortalityE6E7) and WI38 (WI38SV40LT) lung fibroblasts2, respectively, thereby silencing or disrupting the p53 and RB tumour-suppressor pathways11. Checkpoint-deficient cells bypassed senescence and reached crisis at population doubling 105–110 (PD105–110) for IMR90E6E7 and PD85–90 for WI38SV40LT (refs. 2). RNA sequencing (RNA-seq) analysis revealed profound transcriptional changes during crisis with an overlap of upregulated genes  were induced, consistent with an inflammatory status associated with replicative crisis  were introduced into primary human IMR90  entered crisis around PD90 for WI38SV40LT and PD107 for IMR90E6E7, when cell death was frequent and replicative ability was reduced ) contains two N-terminal nucleic-acid-binding domains (Zα1 and Zα2) followed by RHIM domains (RHIM1 and RHIM2) and a structurally undefined C terminus; the short isoform (ZBP1(S)) lacks Zα1 but retains Zα228  or mutant forms of ZBP1(S) 31 did not induce ISGs and cell death  analysis showed enhanced TERRA transcription from critically short telomeres in crisis or telomeres depleted of TRF238 . We first depleted TRF2 in growing IMR90E6E7 fibroblasts expressing WT ZBP1(S) and then performed formaldehyde cross-linking combined with ZBP1-associated RNA immunoprecipitation ) alone or fused to the VP16 transcriptional activation domain (VP16–TRF1(ΔN))ted Fig. . WT ZBP1ted Fig. . Accordited Fig. . No lossted Fig. . Partialted Fig. . Cells eted Fig. . These f43 and MDA544, which translocate to mitochondria to interact and activate MAVS35; however, they often form aggregated structures rather than filaments45, suggesting an alternative oligomerization mechanism for ZBP1. We found co-localization of ZBP1 filaments with a subset of mitochondria based on MOM (TOM20) and matrix (MitoTracker and TFAM) markers , whereas RIG-I and MDA5 activate MAVS through CARD–CARD interactions35. Finally, we propose that TERRA transcripts constitute a structure-specific ligand for ZBP1, with low or no binding affinity to RIG-I and MDA5. These differences may prime ZBP1 to propagate the inflammatory signalling cascade specifically in response to telomere dysfunction, revealing a unique mechanism of MAVS-dependent IFN activation and subsequent cell death by autophagy.Although ZBP1 functions as an RNA sensor with the ability to activate a MAVS and IFN signalling response, it differs from RIG-I and MDA5 in several aspects. First, ZBP1 filament formation occurs at the mitochondrial surface, whereas RIG-IZBP1. However, crisis-associated cell death requires additional downstream activation of ZBP1(S) by TERRA and formation of ZBP1 filaments on mitochondria. This promotes an inflammatory loop leading to the expression of an ISG profile that, in concert with activation of autophagy through an as yet undetermined pathway, drives cell death specifically in replicative crisis. The simultaneous activation of CGAS–DNA-sensing and ZBP1–RNA-sensing pathways enables the innate immune system to orchestrate an efficient, type-I-IFN-dependent cell death response to eliminate precancerous cells with unstable telomeres. These findings highlight a synergy between critically short telomeres, mitochondria and innate immunity that has evolved to prevent age-associated cancer initiation in humans.Our study reveals a mechanism for telomere-mediated tumour suppression, whereby dysfunctional telomeres in crisis stimulate two intertwined cytosolic nucleic-acid-sensing pathways and trigger a lethal IFN response  supplemented with 0.1 mM non-essential amino acids  and 15% fetal bovine serum . Human mammary epithelial cells  were purchased from Lonza and were grown under 5% CO2 and 3% O2 using the mammary epithelial cell medium complete kit . The number of PDs was calculated at each passage using the following equation: PD = log[number collected/seeded number]/log2. Cells have been tested to be free of mycoplasma.IMR90 (CCL-186) and WI38 (AG06814-N) fibroblasts were purchased from ATCC and the Coriell Institute for Medical Research, respectively. IMR90 and WI38 fibroblasts were grown under 7.5% CO52. TRF2 shRNA in pLKO.1 was obtained from Open Biosystems. HPV16 E6E7, SV40 LT and H2B-mCherry in pLXSN3 and hTERT in pBabe were obtained from Karlseder laboratory stock2. To generate shZBP1 1, oligos  were annealed by temperature ramp from 100 °C to 25 °C and cloned in a AgeI- and EcoRI-digested non-coding scramble shRNA vector using T4 DNA ligase. ZBP1 shRNA in pLKO.1 (shZBP1 2) was obtained from Sigma-Aldrich (TRCN0000123050).Non-coding scramble shRNA was obtained from D. Sabatini through Addgene (plasmid 1864)53 and the guide target sequence was cloned as phosphorylated adapter in the Esp3I-digested lentiCRISPRv2 vector. Individual guide RNAs (gRNAs) for the targeting of corresponding genomic exons were designed using the CRISPOR web tool (http://crispor.org). The guide RNA sequences are as follows: sgZBP1 1, 5′-CACCTGGTGCCATTGAAGGG-(PAM)-3′; sgZBP1 2, 5′-GGACGATTTACCGCCCAGGT-(PAM)-3′; sgZBP1 3, 5′-TGGGACACAGCAATGAGATG-(PAM)-3′; sgGFP, 5′-GAAGTTCGAGGGCGACACCC-(PAM)-3′; sgLUC (sgrenilla_luciferase), 5′-GGTATAATACACCGCGCTAC-(PAM)-3′; sgMAVS, 5′-AAGTTACCCCATGCCTGTCC-(PAM)-3′; and sgSTING, 5′-AATATGACCATGCCAGCCCA-(PAM)-3′. ZBP1-knockout clones were generated using sgZBP1 2 5′-GGACGATTTACCGCCCAGGT-(PAM)-3′. After transduction, cells were selected with 1 μg ml−1 puromycin for 1 week and individual clones were isolated using the array dilution method in 96 wells. Clones were tested by western blotting for the absence of ZBP1 protein staining.To generate pooled knockout cell populations and knockout clones lentiCRISPRv2 was used. The plasmid was obtained from F. Zhang through Addgene (plasmid 52961)ZBP1 expression plasmids used here are derivates of pLenti-CMV-GFP-Hygro-(656-4) obtained from E. Campeau and P. Kaufman through Addgene (plasmid 17446)54. The sequence of full-length human ZBP1 transcript variant 1 (GenBank: NM_030776.3) was obtained as gblock from IDT. The empty vector control pLenti-MCS-hygro was cloned in three steps: first, the pLenti-CMV-GFP-Hygro-(656-4) vector was digested with XbaI and SalI and the GFP sequence was replaced with a multiple cloning site (MCS) adapter  to generate pLenti-CMV-MCS-Hygro. Second, the CMV promoter in the pLenti-CMV-MCS-Hygro vector was excised using ClaI and BamHI and replaced with a second adapter . Finally, pLenti-MCS-WPRE-Hygro was digested with SalI and XhoI to remove the WPRE sequence followed by religation to generate pLenti-MCS-Hygro. To clone N-terminally 3×Flag-tagged ZBP1 expression constructs, pLenti-CMV-3×Flag-(GS)5-MCS-Hygro was generated first. For this, the Kozak sequence with a 3×Flag tag and a (GS)5 linker was amplified from pLenti-FNLS-P2A-GFP-PGK-Puro (obtained from L. Dow through Addgene (plasmid 110869)55 using primers 5′-CTAGACCTCAGGATCGCCACCATGGACTATAAGGAC-3′ and 5′-ACGCGTCCCGGGATCCAGAGCCGGACCCGCTCCCGGAGCCCTTATCGTCATCGTCTTTGTAATCAATATCATGAT-3′. The fragment was cloned into BamHI-linearized pLenti-CMV-MCS-Hygro using InFusion cloning .All ZBP1 full-length (ZBP1(L)) and short isoform (ZBP1(S), also known as Delta-exon-2, Delta-Z-alpha or isoform 7) were amplified from full-length ZBP1 gblock DNA using either the primer 5′-GTCCGGCTCTGGATCCATGGCCCAGGCTCCTGCT-3′ or primer 5′-GTCCGGCTCTGGATCCATGGCCCAGGCTCCTGCTGACCCGGGCAGAGAAGCCGAGAGGCCCCAGC-3′ in combination with the primer 5′-ACGCGTCCCGGGATCCCTAAATCCCACCTCCCCACC-3′. ZBP1 fragments were cloned into BamHI-linearized pLenti-CMV-3×Flag-(GS)5-MCS-Hygro using InFusion cloning. To clone C-terminally 3×Flag-tagged ZBP1 expression constructs, pLenti-CMV-MCS-(GS)5-3×Flag-Hygro was generated first. For this, a (GS)5 linker with a 3×Flag tag and STOP codon was amplified from pLenti-FNLS-P2A-GFP-PGK-Puro using the primers 5′-CGACTCTAGAGGATCCGGCTCCGGGAGCGGGTCCGGCTCTGACTATAAGGACCACGACGG-3′ and 5′-GAGGTTGATTGTCGACTCACTTATCGTCATCGTCTTTGTAATC-3′. The fragment was cloned into BamHI- and SalI-digested pLenti-CMV-GFP-Hygro-(656-4) using InFusion cloning.ZBP1 gblock DNA and the pLenti-CMV-3×Flag-(GS)5-ZBP1(S)-Hygro plasmid, respectively, with the primers listed below. Fragments were cloned into BamHI-digested pLenti-CMV-MCS-(GS)5-3×Flag-Hygro using InFusion cloning.ZBP1(L)-, ZBP1(S)- and ZBP1(S)-deletion mutants were PCR amplified from full-length ZBP1 construct cloning were as follows: ZBP1(L) (amino acids 1–429) and ZBP1(S)  ZBP1, 5′-CGACTCTAGAGGATCCATGGCCCAGGCTCCTG-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′; ZBP1(S)-ΔZα2 (amino acids 167–429), 5′-CGACTCTAGAGGATCCATGCCAGAAGATTCTGGAAGAAGAGCAA-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′; ZBP1(S)-ΔZα2-ΔRHIM1-ΔRHIM2 (amino acids 278–429), 5′-CGACTCTAGAGGATCCATGCCGTCCGAGGGCCCT-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′; ZBP1(S)-ΔC , 5′-CGACTCTAGAGGATCCATGGCCCAGGCTCCTG-3′ and 5′-TCCCGGAGCCGGATCCGGCGGCTTCCCCCT-3′; ZBP1(S)-ΔRHIM1  N-terminal fragment, 5′-CGACTCTAGAGGATCCATGGCCCAGGCTCCTG-3′ and 5′-TCCCTGGAGGGTCCATTCTGGCAGATCA-3′ and C-terminal fragment 5′-TGGACCCTCCAGGGAGGACGGT-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′; ZBP1(S)-ΔRHIM2  N-terminal fragment, 5′-CGACTCTAGAGGATCCATGGCCCAGGCTCCTG-3′ and 5′-TCGGACGGCTGGGGCCCCCAGG-3′ and C-terminal fragment, 5′-GCCCCAGCCGTCCGAGGGCCCTGC-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′; and ZBP1(S)-ΔRHIM1-ΔRHIM2  N-terminal fragment, 5′-CGACTCTAGAGGATCCATGGCCCAGGCTCCTG-3′ and 5′-TCGGACGGGGGTCCATTCTGGCAGATCA-3′ and C-terminal fragment, 5′-TGGACCCCCGTCCGAGGGCC-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′. Zα2, RHIM1 and RHIM2 point mutations were introduced in the pLenti-CMV-ZBP1(S)-(GS)5-3×Flag-Hygro vector by site-directed mutagenesis using the QuikChange Lightning Site-Directed Mutagenesis Kit . The primers used here for site-directed mutagenesis were as follows: ZBP1(S)-mZα2 , 5′-GGACAGCAAAAGATGTGGCCCGAGACTTGGCCAGGATGAAGAGCAGGC-3′ and 5′-GCCTGCTCTTCATCCTGGCCAAGTCTCGGGCCACATCTTTTGCTGTCC-3′; ZBP1(S)-mRHIM1 , 5′-TGGATTTCCATTGCAAACTCCGAAGCCGCCGCGGCTGCACACGGGAACATCATTACAAGACAGAC-3′ and 5′-GTCTGTCTTGTAATGATGTTCCCGTGTGCAGCCGCGGCGGCTTCGGAGTTTGCAATGGAAATCCA-3′; and ZBP1(S)-mRHIM2 , 5′-GCAGTCCATACTGAGACGGGCGGCGGCGGCACACAGCAATGAGATGAGGC-3′ and 5′-GCCTCATCTCATTGCTGTGTGCCGCCGCCGCCCGTCTCAGTATGGACTGC-3′.The primers for the C-terminal tagged 56. For this, VP16-TRF1ΔN and TRF1ΔN were PCR amplified from pINDUCER20-VP16–TRF1ΔN(44–439) and pINDUCER20-TRF1ΔN(44–439), respectively (both plasmids were kind gifts from P. M. Lieberman57), using either primer 5′-TGGAGAATTGGCTAGCATGGCCCCCCCGACCG-3′ or primer 5′-TGGAGAATTGGCTAGCATGCTTCTCGAGTGCCAGGTGC-3′ in combination with primer 5′-CCCCAACCCCGGATCCTCAGTCTTCGCTGTCTGAGGAAATCAG-3′. Fragments were cloned into the NheI and BamHI-digested pLPT-Empty vector using InFusion cloning.To express untagged VP16-TRF1ΔN and TRF1ΔN from a doxycycline-inducible tight TRE promoter, VP16-TRF1ΔN and TRF1ΔN was subcloned into pLPT-Empty58 and (GS)5-linker-3×Flag with the primers 5′-CGACTCTAGAGGATCCGGCTCCGGGAGCGGGTCCGGCTCTGACTATAAGGACCACGACGG-3′ and 5′-CACGATGGCCTTATCGTCATCGTCTTTGTAATC-3′ from pLenti-FNLS-P2A-GFP-PGK-Puro5. The two fragments were cloned into BamHI/SalI-digested pLenti-CMV-GFP-Hygro(656-4) (see methods of ref. 4) using InFusion cloning. Next, pLenti-CMV-MCS-(GS)5-3Flag-OMM-hygro was linearized with BamHI and ZBP1(S) that was amplified with the primers 5′-CGACTCTAGAGGATCCATGGCCCAGGCTCCTG-3′ and 5′-TCCCGGAGCCGGATCCAATCCCACCTCCCCACC-3′ from pLenti-CMV-3×Flag-(GS)5-ZBP1(S)-hygro was cloned into the linearized vector using InFusion cloning. Finally, pLenti-CMV-3×Flag-(GS)5-ZBP1(S)-OMM-hygro was generated by InFusion cloning using amplified fragments of pLenti-CMV-ZBP1(S)-(GS)5-3Flag-OMM with the primers 5′-GCCATCGTGGGAGGCATG-3′ and 5′-GGATCCTCTAGAGTCGGTGTCT-3′ and 3×Flag-(GS)5-ZBP1(S) with the primers 5′-GACTCTAGAGGATCCGCCACCATGGACTATAAGGACCACGAC-3′ and 5′-GCCTCCCACGATGGCAATCCCACCTCCCCACC-3′ from pLenti-CMV-3×Flag-(GS)5-ZBP1(S)-hygro.To localize ZBP1(S) to the mitochondrial outer membrane, the outer mitochondrial membrane localization (OMM) signal of FIS1 was fused to the C terminus of 3×Flag-ZBP1(S). The construct was cloned in three steps: first, pLenti-CMV-MCS-3×Flag-OMM-hygro was generated by amplifying the OMM of FIS1 with the primers 5′-CGATAAGGCCATCGTGGGAGGC-3′ and 5′-GAGGTTGATTGTCGACTCAGGATTTGGACTTGGACACAG-3′ from MAVS-Mito (obtained from J. Kagan through Addgene (plasmid 44556)T4 ligase (M0202S), T4 PNK (M0201S) and all restriction enzymes used, except for Esp3I (Thermo Scientific ER0452), were obtained from NEB. All primers were ordered from Eton Bioscience. PCR reactions for cloning were performed using KOD Hot Start DNA Polymerase  according to the manufacturer’s protocol.−1), images were taken using the Revolve fluorescence microscope and analysed with CellProfiler v.4.2.1 using a customized pipeline. Total nuclei and Cytotox-dye-positive nuclei were segmented using an integrated intensity-based object detection module. The percentage of dead cells was calculated by dividing the number of Cytotox-dye-positive nuclei by the total number of nuclei multiplied by 100.For real-time assessment of cell death, 2,000 cells per well per condition in triplicate were seeded in 96-well plates  the day before the experiment. The next day, the medium was changed to medium containing 250 nM IncuCyte Cytotox green dye  and IncuCyte Nuclight Rapid red dye (1:100 dilution) . Cells were seeded in 96-well plates the day before the experiment and the medium changed to medium containing 250 nM IncuCyte Cytotox green dye on the day of the experiment. Cells were imaged with at least two fields per well every 2 h using the ×10 objective on the IncuCyte S3 or IncuCyte Zoom live-cell analysis system (Sartorius). The total number of nuclei and the Cytotox-green-dye-positive nuclei were quantified using the IncuCyte analysis software. The percentage of dead cells was calculated by dividing the Cytotox green dye positive nuclei by the total number of nuclei multiplied by 100. In Extended Data Figs. 56. In brief, HEK293T  cells were transfected with 7 µg of DNA using Lenti-X Packaging Single-Shot system . Then, 48 h after transfection, viral supernatant was collected, supplemented with serum and used for transduction in the presence of Lenti-Blast . To produce retrovirus, Phoenix cells were transfected with 20 µg of DNA using 100 µM of chloroquine. Then, 5 h after transfection, fresh medium was added. The viral supernatant was collected 24 h later and used for transduction in the presence of polybrene 4 µg ml−1. Then, 48 h after infection, cells were washed and selected with 1 µg ml−1 puromycin, 600 µg ml−1 G418, or 90 µg ml−1 hygromycin. IMR90E6E7 and WI38SV40LT fibroblasts were subjected to long-term culturing under antibiotic selection.Lentiviral and retroviral particles were produced by the laboratory. Production of lentivirus was performed as described previouslyDNA transfections were performed using the Lipofectamine 3000 kit  according to the manufacturer’s instructions. siRNA transfections were performed using the Lipofectamine RNAiMAX kit  according to the manufacturer’s instructions.59. In brief, cells were lysed in NuPage LDS sample buffer  at 1 × 104 cells per µl. Proteins were resolved using NuPage Bis-Tris gel electrophoresis  and transferred to nitrocellulose membranes . Secondary antibodies were peroxidase-conjugated anti-mouse IgG  or anti-rabbit IgG . Peroxidase activity was detected using an ECL kit  and the Syngene G-Box imager. Primary antibodies are listed below.Western blots were performed as described previously60. In brief, cells were treated with 0.2 µg ml−1 of Colcemid  for 3 h, collected and incubated with hypotonic solution (75 mM of KCl) for 7 min at 37 °C. The cell suspension was centrifuged, washed in fixative solution , dropped onto superfrost microscope slides  and air dried overnight. For telomere staining, cells were fixed in 4% formaldehyde in PBS for 10 min, dehydrated in a series of ethanol baths  for 3 min each and air-dried for 20 min. Cells were then covered in 0.3 ng µl−1 of the telomeric  PNA probes diluted in hybridization solution , 10 mM Tris pH 7.5). The samples were heated for 5 min at 80 °C, incubated 2 h at room temperature, washed twice in 70% formamide and 10 mM Tris-HCl pH 7.5 and three times in 50 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.08% Tween-20. Slides were finally mounted in ProLong Diamond with DAPI .Metaphase spread preparation was performed as described60. Cells were washed in PBS, fixed in 4% formaldehyde in PBS for 10 min and permeabilized in 0.1% Triton X-100 in PBS for 10 min. For metaphase spreads, cells were treated with 20 ng ml−1 of Colcemid  for 1 h, collected and incubated in hypotonic solution  for 5 min. The cell suspension was cytocentrifuged, fixed in 4% formaldehyde in PBS for 10 min and permeabilized in KCM buffer  for 10 min. In both settings, the samples were incubated in blocking buffer  for 1 h at 37 °C. Cells were incubated with the primary antibody (γH2AX) for 2 h, washed in PBS and incubated with secondary antibody for 1 h at room temperature. The secondary antibodies used were AlexaFluor 568 anti-IgG Mouse  or AlexaFluor 647 anti-IgG Mouse . The samples were finally fixed in 4% formaldehyde in PBS before fluorescence in situ hybridization (FISH) analysis.For interphase nuclei, cells were seeded onto glass coverslips 24 h before the experiment as described previouslyCells were seeded onto glass coverslips 24 h before the experiment. Cells were fixed in 4% paraformadehyde in PBS for 10 min, washed in PBS and incubated in blocking solution of 5% BSA in PBS at room temperature. Cells were then incubated with primary antibodies for 2 h, washed in PBS and incubated with secondary antibodies for 1 h at room temperature. The secondary antibodies used were AlexaFluor 488 goat anti-rabbit IgG (H+L)  and AlexaFluor 568 goat anti-Mouse IgG (H+L) . The samples were finally washed in PBS and mounted in ProLong Diamond with DAPI . For MitoTracker staining, MitoTracker Red CMXRos  was added to the culture medium at a final concentration of 200 nM and incubated for 20 min under tissue culture conditions. After staining, cells were washed twice in prewarmed PBS, then fixed in paraformaldehyde 4% PBS for 10 min and washed in PBS. Imaging was performed using the Zeiss LSM 880 with the Airyscan microscope. ZEN (Zeiss) and ImageJ were used for image analysis.2) and kept at 37 °C for 10 days before fixing with 4% paraformaldehyde in PBS. Cells were then stained with 0.05% crystal violet in distilled water for 20 min.Cells before crisis were seeded at low density , protease inhibitor cocktail , 100 U ml−1 RNasin Ribonuclease Inhibitor ), and incubated for 20 min at 4 °C under slow rotation. The cell lysates were centrifuged at maximum speed at 4 °C for 10 min, and the supernatants were collected and diluted in equal volumes of freshly made fRIP binding/wash buffer , protease inhibitor cocktail , 100 U ml−1 RNasin Ribonuclease Inhibitor ). Diluted lysates were first precleared with Dynabeads Protein G  at a concentration of 25 µl of beads per 5 million cells for 30 min at 4 °C under slow rotation, and then incubated with 10 μg anti-Flag M2 antibodies  previously coupled to 40 μl protein G Dynabeads (per each 5 million cells) for 2 h at 4 °C under slow rotation. The beads were washed twice with fRIP binding/wash buffer and protein–RNA cross-links were reversed by resuspending the beads in 56 μl of RNase-free water + 33 μl of 3× reverse-cross-linking buffer (3× PBS (without Ca2+ and Mg2+), 6% N-lauroyl sarcosine, 30 mM EDTA, 15 mM DTT (added fresh), 10 μl of 20 mg ml−1 proteinase K  and 1 μl of RNasin Ribonuclease Inhibitor. Protein degradation and reverse-cross-linking was performed for 1 h at 42 °C, followed by another hour at 55 °C. RNA was recovered by resuspending the beads and reaction buffer in 1 ml TRIzol Reagent  and purified using the Direct-zol RNA Microprep kit as recommended by manufacturer .fRIP–seq was performed as previously described with minor modificationshttp://www.wistar.org/lab/harold-c-riethman-phd/page/subtelomere-assemblies) using STAR v.2.5.3a allowing up to 101 mapping locations62. Primary alignments were assigned randomly, and each read is represented by one mapping location. Library sizes were normalized to 1 × 107 for comparison and the log2 ratio of enrichment of immunoprecipitates versus input was calculated at each base in the subtelomere using HOMER v.4.10 and the assembly available of human subtelomeres63. Visualizations were performed using R v.3.6.1 (R Core Team). Graphical packages  were used to visualize data.RNA-seq libraries were prepared with immunoprecipitated RNA using the TruSeq stranded total RNA sample preparation kit according to the manufacturer’s protocol (Illumina). RNA-seq libraries were multiplexed, normalized and pooled for sequencing. The libraries were sequenced on the MiniSeq system (Illumina) with paired-end 37 bp reads. Image analysis and base calling were performed using Illumina CASAVA v.1.8.2. on the MiniSeq system and sequenced reads were quality-tested using FASTQC. Read pairs were mapped individually to the most complete assembly available of human subtelomeres . qPCR was performed on the CFX384 Touch Real-Time PCR Detection System (BioRad). Reactions were run in triplicates with Power SYBR Green Master Mix  in a total volume of 10 μl with standard cycling conditions. Relative gene expression was normalized using GAPDH as a housekeeping gene and calculated using the Comparative CT Method (ΔΔCT method). The primers are listed below.Total RNA was isolated using TRIzol  and purified using the RNAeasy Mini Kit  according to the manufacturer’s instructions. Genomic DNA was eliminated by double digestion with DNase I . For RT–qPCR, 3.5 μg of RNA was reverse-transcribed either with random hexamers for measuring ISGs or with 2 μM of 3-primed Klenow labelling of an 800 bp [TTAGGG]n fragment in the presence of [α32P]dCTP. After hybridization, the membrane was washed twice in 2× SSC and 0.1% SDS for 10 min at room temperature and then once for 10 min at 50 °C. The radioactive signal was detected using the Typhoon FLA 9000 imager . After the signal acquisition, membranes were stripped and rehybridized at 50 °C overnight with 32P-dCTP-labelled probes targeting GAPDH transcripts. Signals were measured using ImageJ.Total RNA (10 μg) was blotted onto a positively charged nitrocellulose membrane . For fRIP, 200 ng of RNA from IP or input samples were used. For RNase controls, RNA was incubated with RNase A  at 37 °C for 1 h. RNA was fixed by ultraviolet cross-linking  and TERRA was detected by hybridizing overnight at 55 °C with a Church mix containing telomeric repeat probes generated by [CCCTAA]65. In brief, genomic DNA was isolated by phenol–chloroform extraction and digested with AluI and MboI overnight. A total of 4 µg of digested gDNA was separated on 0.7% agarose gel at 40 V and transferred to a positively charged Nylon membrane . After cross-linking the DNA and prehybridization  for 2 h at 65 °C, the membrane was incubated with digoxigenin-labelled TelG probe diluted in hybridization buffer  overnight at 65 °C. Digoxigenin-labelled TelG probe was generated as previously described66. Then, the membrane was washed three times with wash buffer 1 , one time with wash buffer 2 (2× SSC) for 15 min each and blocked in freshly prepared blocking solution  blocking reagent ) for 30 min. Next, the membrane was incubated for 30 min in anti-digoxigenin-AP antibodies  diluted in blocking solution, washed twice in wash buffer 3  Tween-20) for 15 min each and equilibrated in AP buffer  for 2 min. Digoxigenin-labelled telomeric DNA was detected using CDP-star ready to use  solution.TRF analysis was performed as previously described67. This ready-to-use lentiviral library has 77,441 gRNAs, targeting 19,114 protein-coding genes, with approximately 4 sgRNAs per gene. For adequate representation of each sgRNA, a total of 100 million pre-crisis (PD100) IMR90E6E7 fibroblasts were transduced with the lentiviral library at a multiplicity of infection of 0.5. Transductions were performed in six-well plates (3 million cells per well) in medium containing 4 μg ml−1 polybrene while centrifuging at 1,000 rcf for 1 h at 33 °C (spinfection). The next day, cells were transferred to Cell Factory System , and puromycin-containing medium (1 μg ml−1) was added for 7 days to eliminate uninfected cells and achieve genome-edited cell pools. After selection, cells were pooled together and divided into two technical replicates of 30 million cells each corresponding to the library baseline control at day 0. Another 30 million cells were replated into one Cell Factory System and positive selection was performed by growing cells for additional 15 days, at which point crisis-associated cell death was frequent. Two technical replicates of 30 million cells each were prepared by collecting cells at day 15, and their genomic DNA was extracted using a modified version of QIAGEN’s DNeasy Blood and Tissue Kit provided by the FLI-Seq Library Prep for CRISPR kit (Eclipse Bioinnovations). Genomic DNA was also extracted from the baseline count control sample at day 0. DNA fragments containing the sgRNA sequences were first captured from sheared gDNA, amplified by PCR using the FLI-Seq Library Prep for CRISPR kit (Eclipse Bioinnovations) and processed for next-generation sequencing. CRISPR libraries were multiplexed, normalized and pooled for sequencing. To compensate for low base diversity in CRISPR libraries, high-diversity libraries or PhiX Control v3 Library were spiked in for sequencing and the libraries were sequenced on the HiSeq 2500 system (Illumina) as single reads. Image analysis and base calling were performed using Illumina CASAVA v.1.8.2 on the HiSeq 2500 system and sequenced reads were quality-tested using FASTQC. Fold changes of sgRNA read counts before (day 0) and after (day 15) enrichment were calculated with the PinAPL-Py software and hits were defined as genes with a read count ratio in the survival pool to the baseline control log2[fold change] ≥ 2 in both replicates. GO enrichment analysis was performed using the WEB-based Gene Set Analysis Toolkit . The top 20 GO terms with an FDR value of <0.05 were considered to be statistically significant and visualized using the ClueGO and ggplot2 R packages.The human Brunello CRISPR knockout pooled library was obtained from D. Root and J. Doench through Addgene (73179-LV)68 and mapped to the hg19 human genome using the STAR aligner (v.2.5.3a)62 with the default parameters. Raw or transcripts per kilobase million (TPM) gene expression was quantified across all the exons of RefSeq genes with analyzeRepeats.pl in HOMER (v.4.11.1)63, which used the top-expressed isoform as proxy for gene expression. Differential gene expression was performed on the raw gene counts with the R package, DESeq2 (v.1.24.0)69, using replicates to compute within-group dispersion. Differentially expressed genes were defined as having a FDR < 0.05 and a log2[fold change] > 0.585 (~1.5 fold) when comparing two experimental conditions. GO enrichment analysis was performed using the WEB-based Gene Set Analysis Toolkit”  and the R package clusterProfiler. The top 20 GO terms with an FDR value of <0.05 were considered to be statistically significant and visualized using the ClueGO and ggplot2 R packages. Heat maps and Venn diagrams were generated using the R packages ComplexHeatmap and VennDiagram, respectively.Total RNA was isolated using TRIzol  and purified using the RNAeasy Mini Kit  according to the manufacturer’s instructions. Genomic DNA was eliminated by double digestion with DNase I . The quality of the isolated total RNA was assessed using the Agilent TapeStation 4200 and RNA-seq libraries were prepared with 500 ng total RNA using the TruSeq stranded mRNA sample preparation kit according to the manufacturer’s protocol (Illumina). RNA-seq libraries were multiplexed, normalized and pooled for sequencing. The libraries were sequenced on the HiSeq 4000 system (Illumina) as 50 bp single reads or the NextSeq 500 system (Illumina) as 75 bp single reads. Image analysis and base calling were performed using Illumina CASAVA v.1.8.2 on the HiSeq 4000 system and sequenced reads were quality-tested using FASTQC. Sequenced reads were quality-tested using FASTQC (v.0.11.8)One million cells were seeded in a 10 cm plate for 24 h. Conditioned cell culture medium was collected and centrifuged at 14,000 rpm for 10 min at 4 °C to remove cellular debris. Three technical replicates of 50 μl each were collected for the assay. For preparation of standards, GlutaMax-DMEM  supplemented with 0.1 mM non-essential amino acids  and 15% fetal bovine serum  was used. IFNβ secretion in cell culture supernatant was quantified using the IFNβ enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instruction. Supernatants from the treated cells were collected and incubated in IFNβ ELISA kit for 2 h at room temperature and washed three times with wash buffer. The optical density at 450 nm for each sample was measured with a microplate reader and data obtained were plotted against a four-parameter logistic standard curve to determine the concentration of IFNβ.The reagents were as follows: recombinant human IFN-beta protein: R&D Systems, 8499-IF-010/CF; poly(dA:dT): InvivoGen, tlrl-patn; poly(I:C): InvivoGen, tlrl-pic; 2′,3′-dideoxycytidine: Sigma-Aldrich, D5782; deoxyribonucleic acid sodium salt from herring testes (HT-DNA): Sigma-Aldrich, D6898; MitoTracker: Invitrogen, M22426; IncuCyte Nuclight Rapid red dye: Sartorius, 4717; IncuCyte Cytotox red dye: Sartorius, 4632; IncuCyte Cytotox green dye: Sartorius, 4633; doxycycline: Sigma-Aldrich, D9891; staurosporine: Cell Signaling Technology, 9953; recombinant human TNF-alpha protein: R&D Systems, 210-TA; BV6: Selleckchem, S7597; z-VAD-FMK: Cell Signaling Technology, 60332; LPS: Cell Signaling Technology, 14011; nigericin: Cayman Chemical, 11437; Hoechst 33342: Thermo Fisher Scientific, 62249.t-tests. Multiple comparisons were performed using one-way ANOVA followed by Tukey’s or Dunnett’s multiple-comparisons test. For all representative findings, two or three independent experiments were performed, and similar results were obtained. Significance in all figures is denoted as follows: *P <  0.05, **P < 0.01, ***P < 0.001.Statistical analysis was performed using Prism 9. Comparisons between two groups were performed using unpaired two-tailed Student’s P values are as follows.Exact Figure Figure TRF2 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 1 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 2 (<0.0001). CCL5: shScramble versus shTRF2 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 1 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 2 (<0.0001). CXCL10: shScramble versus shTRF2 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 1 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 2 (<0.0001). IFI44: shScramble versus shTRF2 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 1 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 2 (<0.0001). ISG15-shScramble versus shTRF2 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 1 (<0.0001); shTRF2-siCtrl versus shTRF2-siZBP1 2 (<0.0001).Figure TRF2 (<0.0001). 13q, shScramble versus shTRF2 (<0.0001). 15q, shScramble versus shTRF2 (<0.0001). Xp, shScramble versus shTRF2 (<0.0001). Xq, shScramble versus shTRF2 (0.0015).Figure Figure Figure Figure Figure MAVS (0.9002); Flag–ZBP1(S) siCtrl versus Flag–ZBP1(S) siMAVS (0.3629); Flag–ZBP1(S)–FIS1 siCtrl versus Flag–ZBP1(S)–FIS1 siMAVS (<0.0001). CXCL10: vector siCtrl versus vector siMAVS (0.9537). Flag–ZBP1(S) siCtrl versus Flag–ZBP1(S) siMAVS (0.0001). Flag–ZBP1(S)–FIS1 siCtrl versus Flag–ZBP1(S)–FIS1 siMAVS (<0.0001). IFI44: vector siCtrl versus vector siMAVS (>0.9999). Flag–ZBP1(S) siCtrl versus Flag–ZBP1(S) siMAVS (0.9966). Flag–ZBP1(S)–FIS1 siCtrl versus Flag–ZBP1(S)–FIS1 siMAVS (<0.0001).Figure E6E7, 24 h after incubation with Cytotox. sgGFP versus sgZBP1 1 (<0.0001). sgGFP versus sgZBP1 2 (<0.0001); sgGFP versus sgZBP1 3 (<0.0001); sgLUC versus sgZBP1 1 (<0.0001); sgLUC versus sgZBP1 2 (<0.0001); sgLUC versus sgZBP1 3 (<0.0001). WI38SV40LT, 24 h after incubation with Cytotox. sgGFP versus sgZBP1 1 (<0.0001); sgGFP versus sgZBP1 2 (<0.0001); sgGFP versus sgZBP1 3 (<0.0001); sgLUC versus sgZBP1 1 (<0.0001); sgLUC versus sgZBP1 2 (<0.0001); sgLUC versus sgZBP1 3 (<0.0001).Extended Data Fig. 3d: IMR90E6E7: sgLUC versus sgGFP (0.0668); sgLUC versus sgZBP1 1 (<0.0001); sgLUC versus sgZBP1 2 (<0.0001); sgLUC versus sgZBP1 3 (<0.0001). WI38SV40LT: sgLUC versus sgGFP (0.1293); sgLUC versus sgZBP1 1 (<0.0001); sgLUC versus sgZBP1 2 (<0.0001); sgLUC versus sgZBP1 3 (<0.0001).Extended Data Fig. 3e: IMR90E6E7: sgLUC versus sgGFP (0.9995); sgLUC versus sgZBP1 1 (0.0002); sgLUC versus sgZBP1 2 (<0.0001); sgLUC versus sgZBP1 3 (0.0004). WI38SV40LT: sgLUC versus sgGFP (0.7845); sgLUC versus sgZBP1 1 (0.0018); sgLUC versus sgZBP1 2 (<0.0001). sgLUC versus sgZBP1 3 (0.0011).Extended Data Fig. 3f: IMR90E6E7: growing versus crisis (<0.0001). WI38SV40LT: growing versus crisis (<0.0001).Extended Data Fig. 4b: IMR90IFNAR1 (1.39178 × 10−5); siCtrl versus siIFNAR2 (8.05855 × 10−5); siCtrl versus siCGAS (3.65474 × 10−5); siCtrl versus siAIM2 (0.57384863); siCtrl versus siTLR3 (0.028940902); siCtrl versus siTLR7 (0.130633803); siCtrl versus siTLR9 (0.481604543); siCtrl versus siRIG-I (0.067963804); siCtrl versus siMDA5 (0.078645098); siCtrl versus RIG-I + siMDA5 (0.075426998); siCtrl versus siSTING (0.000445511); siCtrl versus siPYCARD (0.272857261); siCtrl versus siMyD88 (0.16111072); siCtrl versus siTRIF (0.138938302); siCtrl versus siMAVS (3.61752 × 10−5). Experiment 2: siCtrl versus siIFNAR1 (1.48978 × 10−6); siCtrl versus siIFNAR2 (1.4765 × 10−5); siCtrl versus siCGAS (1.40243 × 10−6); siCtrl versus siAIM2 (0.081532291); siCtrl versus siTLR3 (0.244271443); siCtrl versus siTLR7 (0.413559022); siCtrl versus siTLR9 (0.1970414); siCtrl versus siRIG-I (0.015458711); siCtrl versus siMDA5 (0.027374775); siCtrl versus RIG-I + siMDA5 (0.857465154); siCtrl versus siSTING (9.9925 × 10−5); siCtrl versus siPYCARD (0.28638452); siCtrl versus siMyD88 (0.73179998); siCtrl versus siTRIF (0.0188581); siCtrl versus siMAVS (7.6601 × 10−6). Experiment 3: siCtrl versus siIFNAR1 (1.23834 × 10−5); siCtrl versus siIFNAR2 (6.7646 × 10−5); siCtrl versus siCGAS (1.04549 × 10−6); siCtrl versus siAIM2 (0.05401101); siCtrl versus siTLR3 (0.16170163); siCtrl versus siTLR7 (0.01702073); siCtrl versus siTLR9 (0.0189054); siCtrl versus siRIG-I (0.00405314); siCtrl versus siMDA5 (0.373743841); siCtrl versus RIG-I + siMDA5 (0.024471461); siCtrl versus siSTING (0.00368923); siCtrl versus siPYCARD (0.08524613); siCtrl versus siMyD88 (0.14064815); siCtrl versus siTRIF (0.12424426); siCtrl versus siMAVS (0.008386476).Extended Data Fig. 4e: experiment 1: siCtrl versus siExtended Data Fig. 5d: IFNB1: vector versus WT (<0.0001); vector versus ΔZα2 (0.2671); vector versus ΔZα2ΔRHIM1ΔRHIM2 (0.1795); vector versus ΔC (<0.0001); vector versus ΔRHIM1 (0.6671); vector versus ΔRHIM2 (<0.0001); vector versus ΔRHIM1ΔRHIM2 (0.0151). CCL5: vector versus WT (<0.0001); vector versus ΔZα2 (0.0367); vector versus ΔZα2ΔRHIM1ΔRHIM2 (0.978); vector versus ΔC (<0.0001); vector versus ΔRHIM1 (0.0675); vector versus ΔRHIM2 (<0.0001); vector versus ΔRHIM1ΔRHIM2 (0.0816). CXCL10: vector versus WT (<0.0001); vector versus ΔZα2 (0.1945); vector versus ΔZα2ΔRHIM1ΔRHIM2 (0.9998); vector versus ΔC (<0.0001); vector versus ΔRHIM1 (0.1365); vector versus ΔRHIM2 (<0.0001); vector versus ΔRHIM1ΔRHIM2 (0.1988). IFI44: vector versus WT (<0.0001); vector versus ΔZα2 (0.0294); vector versus ΔZα2ΔRHIM1ΔRHIM2 (0.9813); vector versus ΔC (<0.0001); vector versus ΔRHIM1 (0.0287); vector versus ΔRHIM2 (<0.0001); vector versus ΔRHIM1ΔRHIM2 (0.2269). ISG15: vector versus WT (<0.0001); vector versus ΔZα2 (0.0003); vector versus ΔZα2ΔRHIM1ΔRHIM2 (0.0242); vector versus ΔC (<0.0001); vector versus ΔRHIM1 (0.0005); vector versus ΔRHIM2 (<0.0001); vector versus ΔRHIM1ΔRHIM2 (0.0001).ZBP1 (<0.0001); siCtrl versus siRIPK3 (0.9942); siCtrl versus siMLK (>0.9999); siCtrl versus siRIPK1 (>0.9999); siCtrl versus siFADD (0.9425); siCtrl versus siCASP8 (>0.9999); siCtrl versus siNLRP3 (>0.9999); siCtrl versus siPYCARD (>0.9999); siCtrl versus siCASP1 (0.9790); siCtrl versus siIFNAR1 (<0.0001); siCtrl versus siIFNAR2 (<0.0001); siCtrl versus siSTAT1 (<0.0001); siCtrl versus siSTAT2 (<0.0001); siCtrl versus siIRF9 (<0.0001); siCtrl versus siATG5 (<0.0001); siCtrl versus siATG7 (<0.0001); siCtrl versus siATG12 (<0.0001).Extended Data Fig. 6e: vector versus ZBP1(S)–Flag (0.0082); siCtrl versus siTRF2 day 3 (<0.0001); mock versus shTRF2 day 6 (<0.0001); mock versus shTRF2 day 9 (<0.0001); mock versus shTRF2 day 12 (<0.0001).Extended Data Fig. 6g: mock versus shScramble day 3 (0.9934); mock versus shScramble day 6 (0.9996); mock versus shScramble day 9 (0.9994); mock versus shScramble day 12 (0.9996); mock versus shTRF2 day 3 (<0.0001); mock versus shTRF2 day 6 (<0.0001); mock versus shTRF2 day 9 (<0.0001); mock versus shTRF2 day 12 (<0.0001).Extended Data Figure 6h: mock versus shScramble day 3 (>0.9999); mock versus shScramble day 6 (>0.9999); mock versus shScramble day 9 (>0.9999); mock versus shScramble day 12 (>0.9999); mock versus shZBP1 1 (0.9038); siCtrl versus siZBP1 2 (0.2923).Extended Data Figure 6j: siCtrl versus siE6E7: PD42 versus PD45 (>0.9999); PD42 versus PD46 (0.9999); PD42 versus PD51 (0.9999); PD42 versus PD55 (0.9997); PD42 versus PD58 (>0.9999); PD42 versus PD62 (0.9998); PD42 versus PD69 (0.9996); PD42 versus PD73 (>0.9999); PD42 versus PD85 (0.9996); PD42 versus PD87 (0.9998); PD42 versus PD90 (0.9998); PD42 versus PD99 (0.9864); PD42 versus PD100 (0.0891); PD42 versus PD101 (0.0003); PD42 versus PD103 (<0.0001); PD42 versus PD105 (<0.0001); PD42 versus PD106 (<0.0001). IMR90E6E7, hTERT: PD61 versus PD74 (0.8616); PD61 versus PD89 (0.9996); PD61 versus PD95 (0.9995); PD61 versus PD105 (>0.9999); PD61 versus PD108 (0.9882). WI38SV40LT: PD35 versus PD38 (>0.9999); PD35 versus PD41 (>0.9999); PD35 versus PD44 (0.9997); PD35 versus PD47 (>0.9999); PD35 versus PD50 (0.9999); PD35 versus PD53 (>0.9999); PD35 versus PD58 (0.9998); PD35 versus PD62 (0.9998); PD35 versus PD67 (0.9998); PD35 versus PD69 (0.9994); PD35 versus PD71 (0.9997); PD35 versus PD79 (0.9994). PD35 versus PD82 (0.9990); PD42 versus PD86 (<0.0001); PD42 versus PD87 (<0.0001); PD42 versus PD89 (<0.0001); PD42 versus PD90 (<0.0001). WI38SV40LT, hTERT: PD50 versus PD63 (0.9576); PD50 versus PD74 (0.9576); PD50 versus PD77 (0.9796); PD50 versus PD83 (>0.9999); PD50 versus PD92 (0.9576).Extended Data Fig. 7c: IMR90Extended Data Fig. 8a: growing versus crisis (<0.0001).TRF2 (<0.0001).Extended Data Fig. 8b: shScramble versus shExtended Data Fig. 9c: 9p: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (<0.0001). 13q: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (0.0002); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (0.0005). 15q: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (<0.0001). Xp: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (<0.0001). Xq: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (0.0023); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (0.0009).Extended Data Figure 10b: CCL5: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (0.7919). CXCL10–ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (0.9996). IFI44: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(ΔZα2)–Flag TRF1(ΔN) versus ZBP1(S)(ΔZα2)–Flag VP16–TRF1(ΔN) (>0.9999).Extended Data Figure 10d: CCL5: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001); ZBP1(S)(mZα2)–Flag TRF1(ΔN) versus ZBP1(S)(mZα2)–Flag VP16–TRF1(ΔN) (0.8595). CXCL10: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001). ZBP1(S)mZα2–Flag TRF1(ΔN) versus ZBP1(S)(mZα2)–Flag VP16–TRF1(ΔN) (>0.9999). IFI44: ZBP1(S)(WT)–Flag TRF1(ΔN) versus ZBP1(S)(WT)–Flag VP16–TRF1(ΔN) (<0.0001). ZBP1(S)(mZα2)–Flag TRF1(ΔN) versus ZBP1(S)(mZα2)–Flag VP16–TRF1(ΔN) (0.9998).E6E7: TOM20–ZBP1, growing versus crisis (<0.0001); MitoTracker–ZBP1, growing versus crisis (<0.0001); TFAM–ZBP1, growing versus crisis (<0.0001). WI38SV40LT: TOM20–ZBP1, growing versus crisis (<0.0001); MitoTracker–ZBP1, growing versus crisis (<0.0001); TFAM–ZBP1, growing versus crisis (<0.0001).Extended Data Figure 11c: IMR90Extended Data Figure 11d: dsDNA intensity: growing versus crisis (<0.0001); dsRNA intensity: growing versus crisis (<0.0001); number of ZBP1 filaments: growing versus crisis (0.0712).SUV3 (<0.0001); siCtrl versus siPNPase (<0.0001); number of ZBP1 filaments: siCtrl versus siSUV3 (0.6179). siCtrl versus siPNPase (0.3240).Extended Data Figure 11e: dsRNA intensity: siCtrl versus siMAVS (0.6383); siCtrl versus siMDA5 (0.9499); siCtrl versus siRIG-I (>0.9999); siCtrl versus siMDA5 + RIG-I (0.9959). ZBP1(S)(WT)–Flag VP16–TRF1(ΔN): siCtrl versus siMAVS (<0.0001), siCtrl versus siMDA5 (0.2942); siCtrl versus siRIG-I (0.9933); siCtrl versus siMDA5 + RIG-I (0.8552). CXCL10: ZBP1(S)(WT)–Flag TRF1(ΔN): siCtrl versus siMAVS (0.9997); siCtrl versus siMDA5 (0.9980); siCtrl versus siRIG-I (0.9973); siCtrl versus siMDA5 + RIG-I (>0.9999). ZBP1(S)(WT)–Flag VP16–TRF1(ΔN): siCtrl versus siMAVS (<0.0001); siCtrl versus siMDA5 (0.0645); siCtrl versus siRIG-I (0.8284); siCtrl versus siMDA5 + RIG-I (0.1366). IFI44: ZBP1(S)(WT)–Flag TRF1(ΔN): siCtrl versus siMAVS (0.9999); siCtrl versus siMDA5 (>0.9999); siCtrl versus siRIG-I (>0.9999); siCtrl versus siMDA5 + RIG-I (>0.9999). ZBP1(S)(WT)–Flag VP16–TRF1(ΔN): siCtrl versus siMAVS (<0.0001); siCtrl versus siMDA5 (<0.0001); siCtrl versus siRIG-I (<0.0001); siCtrl versus siMDA5 + RIG-I (<0.0001).Extended Data Figure 12c: CCL5: ZBP1(S)(WT)–Flag TRF1(ΔN): siCtrl versus siE6E7: vector siCtrl versus siMAVS (>0.9999); vector siCtrl versus siIFNAR2 (>0.9999); Flag–ZBP1(S)–FIS1 siCtrl versus siMAVS (<0.0001); Flag–ZBP1(S)–FIS1 siCtrl versus siIFNAR2 (<0.0001). HMECs: vector siCtrl versus siMAVS (>0.9999); vector siCtrl versus siIFNAR2 (>0.9999); Flag–ZBP1(S)–FIS1 siCtrl versus siMAVS (<0.0001); Flag–ZBP1(S)–FIS1 siCtrl versus siIFNAR2 (<0.0001).Extended Data Figure 13d: IMR90TRF2, ACAGAAGCAGTGGTCGAATC, pLKO.1, Open Biosystems, TRCN0000018358; shZBP1 1, CCAAGTCCTCTACCGAATGAA, pLKO.1; shZBP1 2, GCACAATCCAATCAACATGAT, pLKO.1, Sigma-Aldrich, TRCN0000123050.shRNAs were as follows: Scramble, CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG, pLKO.1, Addgene, 1864; ZBP1 1 : GGACACGGGAACAUCAUUA; ZBP1 2 : CAAAAGAUGUGAACCGAGA; ZBP1 3 : GGAUUUCCAUUGCAAACUC; ZBP1 4 : CAAAGUCAGCCUCAAUUAU; MB21D1 : GAAGAAACAUGGCGGCUAU, AGGAAGCAACUACGACUAA, AGAACUAGAGUCACCCUAA, CCAAGAAGGCCUGCGCAUU; TMEM173 : UCAUAAACUUUGGAUGCUA, CGAACUCUCUCAAUGGUAU, AGCUGGGACUGCUGUUAAA, GCAGAUGACAGCAGCUUCU; MAVS 1 : GCAAUGUGGAUGUUGUAGA; MAVS 2 : AAGUAUAUCUGCCGCAAUU; MAVS 3 : CAUCCAAAGUGCCUACUAG; MAVS 4 : CAUCCAAAUUGCCCAUCAA; PYCARD : GGAAGGUCCUGACGGAUGA, UCACAAACGUUGAGUGGCU, GGCCUGCACUUUAUAGACC, CCACCAACCCAAGCAAGAU; MYD88 : CGACUGAAGUUGUGUGUGU, GCUAGUGAGCUCAUCGAAA, GCAUAUGCCUGAGCGUUUC, GCACCUGUGUCUGGUCUAU; TLR7 : CAACAACCGGCUUGAUUUA, GGAAAUUGCCCUCGUUGUU, GAAUCUAUCACAAGCAUUU, GGAAUUACUCAUAUGCUAA; TLR3 : GAACUAAAGAUCAUCGAUU, CAGCAUCUGUCUUUAAUAA, AGACCAAUCUCUCAAAUUU, UCACGCAAUUGGAAGAUUA; TLR9 : CAGACUGGGUGUACAACGA, GCAAUGCACUGGGCCAUAU, CGGCAACUGUUAUUACAAG, ACAAUAAGCUGGACCUCUA; TLR8 : CAACGGAAAUCCCGGUAUA, CAGAAUAGCAGGCGUAACA, GUGCAGCAAUCGUCGACUA, CUUCCAAACUUAUCGACUA; DDX58 : GCACAGAAGUGUAUAUUGG, CCACAACACUAGUAAACAA, CGGAUUAGCGACAAAUUUA, UCGAUGAGAUUGAGCAAGA; IFIH1 : GAAUAACCCAUCACUAAUA, GCACGAGGAAUAAUCUUUA, UGACACAAUUCGAAUGAUA, CAAUGAGGCCCUACAAAUU; TICAM1 : GGAGCCACAUGUCAUUUGG, CCAUAGACCACUCAGCUUU, GGACGAACACUCCCAGAUC, CCACUGGCCUCCCUGAUAC; IFNAR1 : GCGAAAGUCUUCUUGAGAU, UGAAACCACUGACUGUAUA, GAAAAUUGGUGUCUAUAGU, GAAGAUAAGGCAAUAGUGA; IFNAR2 : CAGAGGGAAUUTUUAAGAA, GAGUAAACCAGAAGAUUUG, CACCAGAGUUUGAGAUUGU, UCACCUAUAUCAUUGACAA; AIM2 : GCACAGUGGUUUCUUAGAG, UCAGACGAGUUUAAUAUUG, GAAAGUUGAUAAGCAAUAC, GUUCAUAGCACCAUAAAGG; SUPV3L1 : UGGCUAAGCUACCGAUUUA, GUAAGGAUGAUCUACGUAA, CGGUGCAGCUCAUGCGGAU, GGAAAGACUUAUCACGCAA; PNPT1 : GACAGAAGUAGUAUUGUAA, ACAGAAAGAUUAUUGGCUA, GAAUGUAAGUUGUGAGGUA, AAUCAGAGAUACUGGUGUA; RIPK3 : CCACAGGGUUGGUAUAAUC, AACCAGCACUCUCGUAAUG, GCUACGAUGUGGCGGUCAA, GACCGCUCGUUAACAUAUA; ATG7 : CCAACACACUCGAGUCUUU, GAUCUAAAUCUCAAACUGA, GCCCACAGAUGGAGUAGCA, GCCAGAGGAUUCAACAUGA; TERRA 1 : AGGGUUAGGGUUAGGGUUAUU; TERRA 2 : GGGUUAGGGUUAGGGUUAGUU; MLKL : GAGCAACGCAUGCCUGUUU, CAAACUUCCUGGUAACUCA, GAAGGAGCUCUCGCUGUUA, GGAUUUGCAUUGAUGAAAC; RIPK1 : CCACUAGUCUGACGGAUAA, UGAAUGACGUCAACGCAAA, GCACAAAUACGAACUUCAA, GAUGAAAUCCAGUGACUUC); FADD : CAUUUAACGUCAUAUGUGA, GGAGAAGGCUGGCUCGUCA, UGACAGAGCGUGUGCGGGA, GCAUCUACCUCCGAAGCGU); CASP8 : GGACAAAGUUUACCAAAUG, GCCCAAACUUCACAGCAUU, GAUAAUCAACGACUAUGAA, GUCAUGCUCUAUCAGAUUU); NLRP3 : GGAUCAAACUACUCUGUGA, UGCAAGAUCUCUCAGCAAA, GAAGUGGGGUUCAGAUAAU, GCAAGACCAAGACGUGUGA); CASP1 : GGAAGACUCAUUGAAACAUA, GAUGGUAGAGCGCAGAUGC, CCGCAAGGUUCGAUUUUCA, GAGUGACUUUGACAAGAUG); STAT1 : GCACGAUGGGCUCAGCUUU, CUACGAACAUGACCCUAUC, GAACCUGACUUCCAUGCGG, AGAAAGAGCUUGACAGUAA); STAT2 : GGACUGAGUUGCCUGGUUA, GGACUGAGGAUCCAUUAUU, GAGCCCUCCCUGGCAAGUUA, GAUUUGCCCUGUGAUCUGA); IRF9 : GCAGAGACUUGGUCAGGUA, CCACCGAAGUUCCAGGUAA, GCGUGGAGCUCUUCAGAAC, GAAAGUACCAUCAAAGCGA); ATG5 : GGCAUUAUCCAAUUGGUUU, GCAGAACCAUACUAUUUGC, UGACAGAUUUGACCAGUUU, ACAAAGAUGUGCUUCGAGA); ATG12 : GAACACCAAGUUUCACUGU, GCAGUAGAGCGAACACGAA, GGGAAGGACUUACGGAUGU, GGGAUGAACCACAAAGAAA).siRNAs were as follows: non-targeting pool : UGGUUUACAUGUCGACUAA, UGGUUUACAUGUUGUGUGA, UGGUUUACAUGUUUUCUGA, UGGUUUACAUGUUUUCCUA); non-targeting : UGGUUUACAUGUCGACUAA; The primers were as follows: 9p-F, GAGATTCTCCCAAGGCAAGG; 9p-R, ACATGAGGAATGTGGGTGTTAT; 13q-F, CTGCCTGCCTTTGGGATAA; 13q-R, AAACCGTTCTAACTGGTCTCTG; 15q-2-F, CAGCGAGATTCTCCCAAGCTAAG; 15q-2-R, AACCCTAACCACATGAGCAACG; Xq-F, AGCAAGCGGGTCCTGTAGTG; Xq-R, GGTGGAACTTCAGTAATCCGAAA; Xp-F, AAGAACGAAGCTTCCACAGTAT; Xp-R, GGTGGGAGCAGATTAGAGAATAAA; GAPDH-F, AGCCACATCGCTCAGACAC; GAPDH-R, GCCCAATACGACCAAATCC; GAPDH RT, GCCCAATACGACCAAATCC; TERRA RT, CCCTAACCCTAACCCTAACCCTAACCCTAA; ZBP1-F, AACATGCAGCTACAATTCCAGA; ZBP1-R, AGTCTCGGTTCACATCTTTTGC; IFNB1-F, ACGCCGCATTGACCATCTAT; IFNB1-R, GTCTCATTCCAGCCAGTGCT; ISG15-F, CGCAGATCACCCAGAAGATCG; ISG15-R, TTCGTCGCATTTGTCCACCA; IFI44-F, AGCCGTCAGGGATGTACTATAAC; IFI44-R, AGGGAATCATTTGGCTCTGTAGA; CCL5-F, CCAGCAGTCGTCTTTGTCAC; CCL5-R, CTCTGGGTTGGCACACACTT; CXCL10-F, GTGGCATTCAAGGAGTACCTC; CXCL10-R, TGATGGCCTTCGATTCTGGATT.Antibodies used were as follows: TRF2 (Karlseder laboratory), ZBP1 , LC3 , STING , CGAS , γH2AX , Flag , GAPDH , TBK1 , phosphorylated TBK1 , STAT1 , phosphorylated STAT1 , IRF3 , phosphorylated IRF3 , MAVS , TOMM20 , TFAM , dsDNA , dsRNA , phosphorylated ATR , phosphorylated ATM , the apoptosis antibody sampler kit , the necroptosis antibody sampler kit  and the pyroptosis antibody sampler kit .Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-023-05710-8.Supplementary Information2 fold change > 2) of the CRISPR–Cas9 screen. Data represent the log2-transformed fold change of read counts before (day 0) and after enrichment (day 15).Supplementary Figs. 1–6. Supplementary Figs. 1–5 contain the raw, uncropped images of the western blots, southern blots of terminal restriction fragments and RNA dot-blots. The molecular mass markers shown throughout this document are in kDa. Supplementary Fig. 6 shows a list of enriched gRNAs (logReporting SummaryPeer Review File


Oratosquilla nepa) exoskeleton, a leftover generated after processing, was used as a starting material for chitosan (CS) production. CS was extracted with different deacetylation times , termed CS−2, CS−3 and CS−4, respectively, and their characteristics and antimicrobial and film properties with agarose (AG) were investigated. Prolonged deacetylation time increased the degree of deacetylation (DDA: 73.56 ± 0.09–75.56 ± 0.09%), while extraction yield (15.79 ± 0.19–14.13 ± 0.09%), intrinsic viscosity (η: 3.58 ± 0.09–2.97 ± 0.16 dL/g) and average molecular weight (Mν: 1.4 ± 0.05–1.12 ± 0.08 (×106 Da)) decreased (p < 0.05). FTIR spectra of extracted CS were similar to that of commercial CS. Among all the CS samples prepared, CS−3 had the best yield, DDA, Mν and antimicrobial activity. Therefore, it was chosen for the development of composite films with AG at different ratios . As the proportion of AG increased, the tensile strength (29.96 ± 1.80–89.70 ± 5.08 MPa) of the composite films increased, while thickness (0.056 ± 0.012–0.024 ± 0.001 mm), elongation at break (36.52 ± 1.12–25.32 ± 1.23%) and water vapor permeability (3.56 ± 0.10–1.55 ± 0.02 (×10−7 g m m−2 s−1 Pa−1)) decreased (p < 0.05). Moreover, lightness of the films increased and yellowness decreased. CS−3/AG (50/50) composite film exhibited high mechanical and barrier properties and excellent compatibility according to FTIR and SEM analyses. According to these finding, mantis shrimp exoskeleton could be used to produce CS. The developed bio-composite film based on an appropriate ratio (50/50) of CS−3 and AG has potential for being used as food packaging material.Mantis shrimp ( Oratosquilla nepa), a marine crustacean species which is classified as fishery bycatch. They are abundant along the coast in the southern part of Thailand and have become economically important due to their palatability and useability in a variety of cooking. By the year 2021, the total of all species of mantis shrimp caught was 700–800 tons per year, with an estimated value of USD 1.18 million  was calculated using Equations (4)–(6):sp = specific viscosity, ηr = relative viscosity, ηred = reduced viscosity, C = chitosan concentration (g/dL), t = flow time of CS solution, and t0 = flow time of the solvent. The ηred was plotted against CS concentration and [η] was calculated from a linear equation at zero concentration. Intrinsic viscosity [η] and viscosity-average molecular weight :−5, and α = 0.742 as determined for the solvent at 25 °C  and M