The transcriptional regulation of K+ transporter genes is one of the key mechanisms by which plants respond to K+ deficiency. Among the HAK/KUP/KT transporter family, HAK5, a high-affinity K+ transporter, is essential for root K+ uptake under low external K+ conditions. HAK5 expression in the root is highly induced by low external K+ concentration. While the molecular mechanisms of HAK5 regulation have been extensively studied, it remains unclear how plants sense and coordinates K+ uptake and translocation in response to changing environmental conditions. Using skor mutants, which have a defect in root-to-shoot K+ translocation, we have been able to determine how the internal K+ status affects the expression of HAK5. In skor mutant roots, under K+ deficiency, HAK5 expression was lower than in wild-type although the K+ concentration in roots was not significantly different. These results reveal that HAK5 is not only regulated by external K+ conditions but it is also regulated by internal K+ levels, which is in agreement with recent findings. Additionally, HAK5 plays a major role in the uptake of Cs+ in roots. Therefore, studying Cs+ in roots and having more detailed information about its uptake and translocation in the plant would be valuable. Radioactive tracing experiments revealed not only a reduction in the uptake of 137Cs+ and 42K+in skor mutants compared to wild-type but also a different distribution of 137Cs+ and 42K+ in tissues. In order to gain insight into the translocation, accumulation, and repartitioning of both K+ and Cs+ in plants, long-term treatment and split root experiments were conducted with the stable isotopes 133Cs+ and 85Rb+. Finally, our findings show that the K+ distribution in plant tissues regulates root uptake of K+ and Cs+ similarly, depending on HAK5; however, the translocation and accumulation of the two elements are different.Potassium (K Anne-Alienor Very . skor3-1 (GK391G12) in Col ecotype was obtained from the NASC (Nottingham Arabidopsis Stock Centre). hak5-1 (SALK_014177) in Col ecotype was obtained from the ABRC . T-DNA insertion and homozygous lines were identified by PCR using primers T-DNA left border primer and a gene-specific primer (Arabidopsis (in vitro on a Petri dish plate with a solid medium containing low-K+ (KCl 10 µM) and high-K+ (KCl 1000 µM) for 7 days. Solid medium is composed of nutrient solution 2, 0.5 mM H3PO4, 9.25 µM H3BO3, 3.6 µM MnSO4, 3 µM ZnSO4, 0.785 µM CuSO4, 0.074 µM NH4Mo7O24, 3.5 mM MES, pH 5.8), 0.5% (w/v) Suc, and 0.8% (w/v) agar). For phenotype assay with Cs+, seeds were sown in vitro on a Petri plate with a solid medium containing low-K+ (KCl 10 µM) nutrient solution. Two days after germination, seedlings were transferred to a liquid medium KCl 10 µM with CsCl 0 or 100 µM for 4 days. For the radiotracer experiment, plants were grown for 14 days in solid medium low-K+ (KCl 10 µM) and high-K+ (KCl 1000 µM) containing nutrient solution. For measurements of K+ distribution phenotype between lines and qRT-PCR experiments, plants were grown in a solid medium containing 100 µM K+ and nutrient solution mix for 12 days, then transferred to sand culture with nutrient solution containing 100 µM K+ for 7 days, then transferred to hydroponic cultures with nutrient solution containing 0, 10, 100, and 1000 µM K+ for 4 days. For cold Cs+ and Rb+ trace experiments, plants were grown in a solid medium containing 100 µM K+ and nutrient solution for 12 days, then transferred to sand culture with nutrient solution containing 100 µM K+ for 10 days, then transferred to hydroponic cultures using a nutrient solution containing low-K+ (KCl 10 µM) and high-K+ (KCl 3000 µM) for 8 days, and then was transferred to a nutrient solution containing 10 µM K+ (12 plants per 15 L nutrient solution containing 10 µM K+) with 1 µM CsCl and 1 µM RbCl for 3 days. Plants were grown in a growth chamber set to 23°C and 8 h Light/16 h dark cycle.For root phenotype assay, seeds were surface sterilized and sown 2.2Pictures of the plates were taken with a camera to analyze lateral root density and root lengths. Root length was measured using the plugin NeuronJ  for the 2.33 (concentrated) at 80°C overnight. After filtration, acid solutions were diluted with 1% HNO3. K+ concentrations in the solution were determined by ICP-OES (Agilent Technology 5800). Cs+ and Rb+ were determined by ICP-MS .The roots of plants were washed in sterile distilled water, separated into shoots and roots, and dried for 3 days at 50°C. Dried samples were digested in HNO2.4°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. For each pair of primers, the PCR efficiency was around 100% and a threshold value was determined. The specificity of PCR amplification was examined by monitoring the presence of the single peak in the melting curves after RT–qPCRs. The relative expression of the gene in each sample was compared to the control sample and was calculated with the delta delta Ct (Ct) method using the following equation: relative expression = 2–ΔΔCt, with ΔCt = Ctsample – Ctcontrol and with Ct = Cttarget gene – Cthousekeeping gene, where Ct refers to the threshold cycle determined for each gene in the exponential phase of PCR amplification (ROC3 gene (At2g16600) . cDNA was synthesized with the qScript cDNA SuperMix (Quanta). Quantitative real-time PCR was conducted with the Light Cycler 480 SYBR Green I Master PCR Mater (Roche) on a Light Cycler 480 (Roche) following the manufacturer’s protocols. The amplification reactions were performed in a total volume of 5 µl, which contained 2 µl cDNA, 2.5 µl SYBR Green premix, and 0.5 µl forward and reverse primers (1µM). The PCR was programmed as follows: 90fication . Using t2g16600) . The sub2.5+ (KCl 10 µM) or high-K+ (KCl 1000 µM) containing nutrient solution were incubated for 2 hours in hydroponic cultures using a nutrient solution containing 1 µM 39K+, 1 µM 133Cs, and 100 Bq/ml 137Cs or a mixed tracer of 42K and 43K 500 Bq/ml. At the end of the uptake period, plant roots were washed with 1 mM KCl and 1 mM CsCl solution. The radioactivity in plants was measured with a gamma counter . For the gamma-ray measurements of the 42K  and 43K  RI mixture, only the spectrum of 43K was measured during the experiments due to its longer half-life.Plants 14 days old grown in solid medium-low-K2.6+ and Rb+. Then, the roots were washed in distilled water and separated into two parts. The two root parts were maintained in the same nutrient solution mix containing low -K+ (KCl 10 µM) for 8 days. The tracer experiment was conducted by placing two root parts in separate pots. Then, 1 µM CsCl or 1 µM RbCl was added to one part of the root for 3 days. Mineralization and measurement are the same as the method described in the ICP analysis.Plants were grown under the same conditions as those used for the cold trace experiments of Cs2.7https://www.arabidopsis.org/index.jsp) under the following accession numbers: At3g02850 for SKOR, At4g13420 for HAK5, At2g30070 for AtKUP1, At2g40540 for AtKUP2, At3g02050 for AtKUP3, At4g23640 for AtKUP4, At4g33530 for AtKUP5, At1g70300 for AtKUP6, At5g09400 for AtKUP7, At5g14880 for AtKUP8, At4g18860 for AtKUP9, At1g31120 for AtKUP10, At2g35060 for AtKUP11, and At1g60160 for AtKUP12.Sequence data for the genes described in this article are in the Arabidopsis TAIR database (33.1skor mutants under different K+ conditions. This study used two independent knockout mutants of the highly selective outward-rectifying K+ channel SKOR including skor1-1 in the WS background (skor3-1 in the Col background. As shown in skor mutants showed similar root architecture to the wild type both under high (1000 µM) and low (10µM) K+ conditions. In contrast, in the hak5-1 mutant, primary root growth is strongly impaired and a significant increase in lateral root density is observed under low K+ conditions (10 µM) (skor mutants is not modified by low K+ conditions compared to hak5-1.We determined the root growth of ckground  and skor (10 µM) Figure 1 (10 µM) . These r3.2+ concentration, plants were grown under different K+ conditions, ranging from 10 µM to 1000 µM. Among all members of the family, only HAK5 is highly induced in roots when the external K+ concentration is less than 100µM, and no expression is detected above this concentration  and the wild-type WS roots . This linear regression, however, is lost in both skor mutants  and skor1-1 . Overall, these results suggest that in addition to external K+ conditions, K+ root concentrations tightly regulate HAK5 expression in roots.To determine and compare the transcriptional regulation of the KUP/KT/HAK transporters in response to Kntration 2. Previntration . In cont3.3+ uptake in Arabidopsis, we examined Cs+ tolerance in both skor and hak5 mutants and their corresponding wild types. Seedlings of wild-type (WS and Col), skor, and hak5-1 knockout mutants were grown in hydroponic cultures for 4 days in the presence of CsCl (100 µM). The skor and hak5 mutants were more resistant to Cs+ toxicity than wild-type plants, as shown in +, whereas the cotyledons of skor1-1, skor3-1, and hak5-1 remained green. As expected, and previously observed, the hak5-1 mutant is less susceptible to Cs+ toxicity due to its reduced uptake of Cs+ . However, under high potassium conditions, there was no difference between wild-type and skor mutants in Cs+ uptake  were performed using 133Cs+ and 85Rb+ since 42K+ tracer has a short half-life of 12.4 hours and so cannot be used for long-term experiments. The roots and shoots of 35-day-old plants were collected separately after 3 days of treatment with 133Cs+ and 85Rb+. Plants grown in high K+ accumulate significantly less 133Cs+ and 85Rb+ than plants grown in low K+ , indicating that both Cs+ and K+ circulate within the plant. In the skor3-1 mutant, there is a slight reduction in the total uptake of Rb+ or Cs+. In addition, less Rb+ was transported to the skor3-1 shoot, as previously observed, whereas it was similarly transferred to the untreated root as in the wild type. However, Cs+ transported to the shoots of skor3-1 was close to that of the wild type, whereas Cs+ transferred from the shoots to the untreated roots was reduced compared to the wild-type plants. In contrast, in the hak5-1 mutant, despite a slight decrease in the total Rb+ absorption, the Cs+ uptake has been dramatically reduced. In the presence of Rb+, hak5-1 plants exhibit broadly comparable distributions (%) to wild-type plants; however, this distribution is significantly altered when Cs+ is applied. In line with previous experiments, the percentage of Cs+ transported from the treated roots to the shoots was dramatically increased (70%) compared to the wild type (48%), but the percentage transported from the shoots to the untreated roots remained unchanged. In the present study, we found that when plants are exposed to low K+ conditions, HAK5 plays a critical role in the uptake and distribution of Cs+ and that SKOR participates in the distribution of Cs+ from the shoot to the roots.There is a constant movement of potassium through the tissues and organs of plants, which is transported in both directions at the same time, upstream by the xylem and downstream by the phloem. Therefore, split root experiments were performed to determine the impact of Kach part Figure 54+ channels and transporters have already been identified in Arabidopsis thaliana , hak5, and skor T-DNA insertion lines to various K+, Cs+, and Rb+ conditions.Proper control of ion uptake is one of the essential elements for plant growth. It is nevertheless important to note that the regulation of ion transport is rather complex, particularly as a result of the redundancy of genes. For example, 71 Kthaliana  and divithaliana . K+ is acterized , and thecterized . Compare of SKOR  and NRT1 of SKOR . Neverthlability . During 4.1+ loading. There is, however, little understanding of the molecular mechanisms involved in the coordination of these two processes. In this study, a correlation was found between the level of HAK5 expression and the concentration of K+ measured in roots. When the concentration of K+ in roots decreases, the expression of HAK5 increases  proteins, ABCG37 and ABCG33, have been identified as high-affinity Cs+ transporters which are K+-independent .A difference in eriments Figure 5ependent . Howeverhe xylem . It is k unknown . On the n shoots . KT/HAK/n shoots . However+ and Cs+ transport, which may contribute to increased fertilizer efficiency and application for phytoremediation. Furthermore, our results indicate that modifying only the surface transporters in the root is not sufficient to improve fertilizer efficiency or facilitate phytoremediation. Further research is required to understand the regulatory mechanism of root surfaces and internal transporters in coordination with each other, which is crucial for the above applications.Finally, we present new insights regarding long-distance KThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/NL and AV designed the research. NL, SK, LM, NV, SC, TN, and JF performed the research. NL and SK analyzed the data and wrote the paper with the contribution of all authors. All authors contributed to the article and approved the submitted version.


On that basis, high efficacy and low toxicity win-win can be obtained. In this study, hollow manganese dioxide nanoparticles (MnO2 NPs) were prepared based on the template method. CDDP was loaded into the hollow cavity and then modified with polydopamine (PDA) and Cy5.5, with the aim of obtaining the nano-drug loading system MnO2/CDDP@PDA-Cy5.5 NPs. The NPs precisely delivered drugs by intelligently responding to the tumor microenvironment (TME). As indicated by the release curves, the NPs release CDDP rapidly by inducing the decomposition of PDA and MnO2 under acidic or redox conditions, and Magnetic resonance imaging (MRI) contrast agent Mn2+ was generated. The results of the in vivo MRI studies suggested that T1 contrast at the tumor site was notably enhanced under the Enhanced permeability and retention (EPR) effect. After the intravenous administration, the effective tumor accumulation exhibited by the NPs was confirmed by magnetic resonance imaging as a function of time. Compared with free CDDP, the in vivo therapeutic effect was remarkably increased. As indicated by the above-described results, MnO2/CDDP@PDA-Cy5.5 NPs is a drug delivery system exhibiting diagnostic and therapeutic functions.The nano drug delivery system MnO Thyroid cancer (TC) refers to a malignant tumor originating from the follicular epithelium or parafollicular epithelial cells of the thyroid gland. It has been confirmed as the most common endocrine malignancy worldwide . This caThe existing treatment method in terms of thyroid cancer comprises surgical removal of the thyroid gland or radiotherapy, or a combination of surgery and radiotherapy . Chemothin vivo is prolonged, its distribution in tumor sites is increased, and the off-target distribution of platinum is reduced, thus reducing the toxic and side effects of cisplatin on the body to some extent  and H+ present in TME to generate paramagnetic Mn2+, such that the contrast of T1 magnetic resonance imaging (MRI) can be significantly improved  and large cavities have been demonstrated to be excellent drug loading/delivery systems to load high quantities of therapeutic agents, whose release may be precisely controlled by tuning the shell structures or coatings  under the EPR effect, such that CDDP was released into the acidic environment. Subsequently, the exposed MnO2 nuclei in tumor cells reacted with excessive H+ to liberate Mn2+, and the ensuing Mn2+ degradation exhibited noticeable MRI characteristics. Thus, the developed nano drug delivery system with integrated diagnosis and treatment can take on critical significance in improving the treatment plan of thyroid cancer and increasing the cure rate of thyroid cancer.A multifunctional nanoparticle was reasonably designed in this study for the first time. The chemotherapy-drug CDDP was encapsulated by MnO3·H2O, ∼28% NH3 in water) and Anhydrous ethanol originated from Shanghai Maclin Biochemical Technology Co., LTD. Phosphate buffer solution (PBS) and Trimethylol aminomethane hydrochloric acid (Tris-HCl) were purchased from Beijing Coolaber Technology Co., LTD. Moreover, potassium permanganate (KMnO4) and sodium carbonate (Na2CO3) were offered by Henan Yaoye Chemical Products Co., LTD. Amino Cy5.5 (NH2-Cy5.5) was purchased from Solarbio Biotechnology Co., LTD. The Millipore system  was used to purify deionized water (H2O). All other chemicals were received in good time and were of reagent quality.Tetraethyl orthosilicate (TEOS), dimethyl sulfoxide (DMSO), CDDP, Dopamine hydrochloride (DA), ammonium hydroxide solution (NH2) nanoparticles were synthesized. 120 ml anhydrous ethanol, 20 ml deionized water, and 10 ml ammonia were introduced into a 250 ml round bottomed flask in turn. After sealing, the sample was subjected to magnetic stirring for 15 min, and then 4.5 mL ethyl orthosilicate was slowly introduced into the mixed solution in the stirring. After sealing, the stirring continued for 8 h at ambient temperature to obtain SiO2 NPs. The resulting SiO2 NPs were washed 3 times with anhydrous ethanol and deionized water, respectively. Subsequently, the NPs were stored in deionized water for the next synthesis SiO2@MnO2.First, solid silica  and then stirred at ambient temperature overnight. The reaction solution was centrifuged at 11,000 rpm/min, the supernatant was discarded, and the obtained precipitates were washed with anhydrous ethanol and deionized water for three times, respectively. On that basis, SiO2@MnO2 nanospheres with core-shell structure were generated.600 mg KMnO2@MnO2 nanoparticles were dissolved into Na2CO3 solution for 12 h at 60°C. Subsequently, the etched dispersion was centrifuged, the supernatant was discarded, and the obtained precipitates were washed with anhydrous ethanol and deionized water for three times in turn to obtain MnO2 NPs.Lastly, core-shell SiO2 was introduced to 10 mL DMSO containing 10 mg CDDP and stir in the dark for 12 h. Subsequently, the remaining CDDP was centrifuged out of the solution to generate MnO2/CDDP NPs. The NPs were dispersed in 10 mL Tris-HCl buffer  containing 10 mg DA, and stirred in the dark for 6 h at ambient temperature to obtain MnO2/CDDP NPs coated with PDA. The final product MnO2/CDDP @PDA NPs was centrifuged and lyophilized for 48 h.10 mg MnO2/CDDP@PDA NPs and 1 mg NH2-Cy5.5 were fully dispersed in 10 ml Tris-HCl buffer and reacted at ambient temperature for 6 h away from light. The mixture was then centrifuged for 10 min at 11,000 rpm/min and washed 3 times with deionized water to remove the remaining NH2-Cy5.5 to obtain MnO2/CDDP@PDA- Cy5.5 NPs. The finished product was freeze-dried for 48 h.10 mg MnO2 and MnO2/CDDP@PDA- Cy5.5 NPs solutions with ultra-pure water and normal saline as the solvents were placed in Xilin bottles, respectively. After the samples stood for 24 h, photos were taken, such that the stability can be compared and analyzed. The morphology, structure and elemental composition of the nanoparticles were characterized through transmission electron microscopy  and X-ray energy dispersion spectroscopy . The nanoparticle size and zeta potential were characterized using the Marvin Nanoparticle Size Analyzer (Nano-ZS90). The valence states of the elements were detected in nanoparticles through X-ray photoelectron spectroscopy . A specific surface area and pore size were investigated using the Barrett–Joyner–Halenda (BJH) method and the Brunauer–Emmett–Teller (BET) method (Micromeritics Instrument Corp. ASAP2460). Ultraviolet-visible spectra of nanoparticles were examined using ultraviolet spectrophotometer .The identical volume of MnO2 loading. Using the method developed previously  and encapsulation efficiency (EE%) of CDDP was examined indirectly by measuring the amount of CDDP in the supernatant, as expressed below:The content of CDDP was determined by UV-vis to evaluate the CDDP of MnOeviously , 5,400 μeviously . After C2/CDDP@PDA (1 mg/mL) solution was dissolved into the dialysis bag, and the dialysis bag was placed in the above beakers containing an appropriate number of media and placed on the magnetic stirrers . At 1, 2, 4, 8, 12, and 24 h, 0.5 mL of the solution outside the dialysis bag was removed, and the corresponding release medium of equal volume was introduced. The Mn2+ content was analyzed by inductively coupled plasma atomic emission spectrometry (ICP-OES). The content of CDDP was determined using an ultraviolet spectrophotometer, and the CDDP release rate was calculated in accordance with the established standard curve and reference formula. Cn denotes the drug concentration in the release medium at the n time point, V represents the total volume of the release medium, Ck expresses the drug concentration in the release medium at the k time point, and V0 is the sampling volume.With different solutions as the release medium, 2 mL of MnO2+ was determined as 90.788 mg/L by ICP-OES to determine the relaxation rate of MnO2/CDDP@PDA-Cy5.5 and assess the MR imaging capabilities of nanoparticles. Next, MnO2/CDDP@PDA-Cy5.5 was dispersed in different conditions. After a 4-h incubation, the MRI study was conducted with a 3.0 T magnetic resonance (MR) device. The T1 value of MnO2/CDDP@PDA-Cy5.5 under different conditions is determined as a function of concentration. The following acquisition parameters were used in T1-weighted image acquisitions: field of view = 90 × 90 mm2, matrix = 268 × 153, slice thickness = 2.0 mm, repetition time = 3,000 ms, echo time = 80 ms.The concentration of Mn2. Nthy-ori3-1 human thyroid normal cells were purchased from Zhaoqing Sea Star Biotechnology Co., LTD., and then cultured in RPMI-1640 supplemented with 10% fetal bovine serum (Corning), 10,000 U/mL penicillin, as well as 10 mg/mL streptomycin (Corning).8305C human thyroid cancer cells originated from Wuhan Procell Life Science and Technology Co., Ltd., and cultured in MEM (including NEAA) supplemented with 10% fetal bovine serum (Corning), 10,000 U/mL penicillin and 10 mg/mL streptomycin (Corning) in a humidified environment of 37°C and 5% CO2/CDDP@PDA-Cy5.5 nanoparticles and then imaged by laser confocal scanning microscopy (CLSM) . Hoechst was used to identify the nuclei, which were then incubated without light for 30 min. The cells were then given three PBS rinses before 1 mL of PBS buffer was added for CLSM imaging.To examine the cell internalization behaviour of various formulations, 8305C cells were incubated with Cy5.5 and MnOy (CLSM) . 8305C c2@PDA-Cy5.5 NPs, 8305C and Nthy-ori3-1 cells were inoculated in 96-well plates at a density of 2×105/well for 12 h. Next, MnO2@PDA-Cy5.5 NPs dispersion in culture medium with different concentrations (Based on the concentration of MnO2) was introduced to the wells. 10 mL of CCK-8 reagent was added to the relevant well after 24 h. After a light shake, the plates were put back under usual conditions for cell incubation for 0.5–1 h. The microplate reader found the OD value at 450 nm after 0.5 h. Cell viability was calculated using the following formula:To assess the cytotoxicity of MnO2/CDDP@PDA-Cy5.5  were incubated with 8305C cells for 24 h, and cell viability was detected by CCK-8 assay. The methods of determination and data processing were the same as those described in the cytotoxicity tests.In addition, nanoparticles carrying CDDP were used to evaluate the therapeutic effect. In this process, free CDDP, MnO5/mL were inoculated into a 6-well plate to assess the effect of MnO2/CDDP@PDA-Cy5.5 on apoptosis of cancer cells. After 12 h incubation, MnO2/CDDP@PDA-Cy5.5, MnO2, free CDDP, and PBS (blank group) were introduced to the respective well and incubated for another 48 h. The tests conformed to the procedure described in the apoptosis kit. Apoptosis was characterized by flow cytometry.8305C cells with a density of 2×105/mL and then subcutaneously injected into the right armpit of naked mice. Drug therapy and imaging studies were conducted on the tumor-bearing mice. Weight and tumor volume were examined every 1 day and detailed records were made.Both BALB/c Nude female  and male SD rats originated from Liaoning Changsheng Biotechnology Co., LTD. All animals were used in investigations following a protocol endorsed by the Qiqihar Medical College’s Experimental Animal Ethics Committee. All experimental nude mice were raised in the SPF environment of Medical College with consistent and suitable alternating light and shade, temperature (22°C ± 1°C) and humidity (50%–60%), during which they were fed and drinking water adequately. 8305C cells at logarithmic growth stage were digested with 0.25% trypsin. The cells were collected after centrifugation and resuspended with normal saline. The cell suspension was prepared at a concentration of 2×102/CDDP@PDA-Cy5.5 injection.The Caliper IVIS Spectrum bioluminescence imaging system was employed for fluorescence imaging of tumor-bearing mice. The anesthetized nude mice were placed in a small animal fluorescence imager, and the internal anesthetic gas release device was directed at the mouth and nose of the nude mice for continuous anesthesia. Fluorescence scanning was performed at 1 h, 2 h, 4 h and 12 h before and after MnO1 signal scanning. After scanning, MnO2/CDDP@PDA-Cy5.5 NPs were injected into nude mice by tail vein at a dose of 5 mg/kg. After injecting NPs for 2 h, 4 h and 8 h, the nude mice were scanned for T1 signal again, and the results were recorded and then analyzed. The following acquisition parameters were used in T1-weighted image acquisitions: field of view = 90 × 90 mm2, matrix = 268 × 153, slice thickness = 2.0 mm, repetition time = 3,000 ms, echo time = 80 ms.After anesthesia, nude mice were placed on a 3.0 T Philips MRI bed for T3. Mice were randomly divided into four groups with three mice in the respective group. The four groups comprised the PBS group, the MnO2 group, the CDDP group, and the MnO2/CDDP@PDA-Cy5.5 group. The dose of MnO2/CDDP@PDA-Cy5.5 was 3 mg/kg (based on the measurement of CDDP), and the dose of MnO2 was 30 mg/kg. The drug was injected into the tail vein of nude mice, once every other day, for a total of 7 times. Before each administration, the weight and tumor volume of mice were examined with balance and vernier caliper, and the health status of mice was observed. The equation for calculating tumor size is V = 0.5×L×S2, where V is the volume of the tumor, and L and S are the longest and shortest diameters of the tumor respectively.Treatment is initiated when the tumor has grown to 80–100 mm2+ content was examined by ICP-AES for biological distribution study  of the respective group were extracted, fixed with 4% paraformaldehyde solution, embedded in paraffin, staining with hematoxylin and eosin (H&E), and imaging with digital microscope (HAMAMATSU S210 microscope). After that, UTP notch end labeling (TUNEL) was used to dye the tumor tissue in order to better determine the tumor’s level of apoptosis. Tumors and major organs  of the probe group were extracted and dissolved into aqua regia for 48h, and Pton study .2/CDDP@PDA-Cy5.5NPs, and Saline treated rats were assigned into controls. To obtain serum, blood samples were centrifuged at 4,000 rpm for 10 min at 4 C. Subsequently, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), and urea nitrogen (BUN) concentrations were measured in the serum with an automatic biochemical analysis .Biochemical analysis was conducted on 1.5 mL of blood from SD rats administrated with MnOp > 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001.Data are presented as mean ± standard deviation (SD). The Student's t-test was used for the statistical analysis. The indicators of significance were #2/CDDP@PDA-Cy5.5 as well as the mechanism of diagnosis and treatment. In brief, hollow mesoporous MnO2 nanoparticles were first prepared by template etching and ultrasonic cavitation. The chemotherapeutic CDDP was then physically encapsulated in a hollow cavity of manganese dioxide nanoparticles to alleviate its inherent toxicity. To precisely control the release of CDDP, PDA shells containing many active groups were formed on MnO2 NPs by self-polymerization of dopamine monomers under alkaline conditions to form MnO2/CDDP@PDA NPs. In mildly acidic conditions, the PDA shells would depolymerize and release CDDP. Lastly, NH2-Cy5.5 is attached to the surface of MnO2/CDDP@PDA NPs to form MnO2/CDDP@PDA-Cy5.5 NPs through the reaction of amino groups and active esters under weak base conditions.1-A2), manganese dioxide nanoparticles are unstable in ultra-pure water and normal saline, and obvious condensation occurs after 24 h of placement. As can be seen from the comparison before and after 3-A4), the stability of modified MnO2 nanoparticles is significantly improved. In addition, the particle size variation of MnO2/CDDP@PDA-Cy5.5 NPs in pure water and PBS solution was also studied. As shown in 2/CDDP@PDA-Cy5.5 NPs in pure water and PBS has not changed significantly for seven consecutive days, indicating that it has good stability.As can be seen from the comparison before and after 2 NPs and core-shell MnO2/CDDP@PDA-Cy5.5 NPs. All NPs had strong monodispersity and were generally spherical , (131), (300), (160), and (003), respectively, and corresponded to the MnO2 crystal type (JCPDS 14–0644). The above results suggested that the obtained MnO2 displayed a cubic crystal structure , but released slowly in neutral PBS. Compared with the slow drug-release profiles of MnO2/CDDP@PDA at pH 7.4, the release speeds of both CDDP and Mn2+ were found to be faster in weak redox environment. The above-mentioned significant pH and redox-responsive drug release behaviour led to the decreased CDDP release in a neutral environment, prolonged the blood circulation in the body, and facilitated the CDDP release of free pharmaceuticals in times, such that negative side effects on healthy tissues were reduced. Degradation of MnO2/CDDP@PDA in acidic solution (pH 5.5) was also observed by TEM , the signal intensity of T1-weighted MnO2/CDDP@PDA-Cy5.5 NPs with different concentrations was notably enhanced, and the relaxation rate were 15.519 and 14.313 mM−1S−1. The possible reason for the above results may be the responsive degradation of MnO2/CDDP@PDA-Cy5.5 NPs. Under acidic and redox conditions, MnO2/CDDP@PDA-Cy5.5 NPs is reduced to Mn2+, which has MRI T1-weighted imaging capability.As depicted in 2/CDDP@PDA-Cy5.5 (Mn = 1 mM) NPs were treated with different concentrations of GSH at pH = 7.4 and imaged under 3.0 T MR. It can be clearly seen from To evaluate whether the nanoparticles would still display redox responsive MR switchable properties after addition of biologically relevant concentrations of reducing agents, MnO2/CDDP@PDA-Cy5.5 and its intracellular behaviour were observed through CLSM in depth. As depicted in 2/CDDP@PDA-Cy5.5 are all represented by red. Notably, MnO2/CDDP@PDA-Cy5.5 exhibited greater fluorescence intensity in the cytoplasm. Fluorescence pictures of 8305C cells treated with either MnO2/CDDP@PDA-Cy5.5 or free Cy 5.5 were reconstructed in 2.5 dimensions by ZEN 2012 software by utilizing the “optical sectioning” capability of CLSM. Compared with free Cy5.5, MnO2/CDDP@PDA-Cy5.5 entered the cell more, especially with stronger red and blue fluorescence, as evidenced by the presence of a fluorescence signal along the z-axis. In order to further study the uptake of MnO2/CDDP@PDA-Cy5.5 by 8305C cells, we carried out fluorescence quantification by flow cytometry. As shown in 2/CDDP@PDA-Cy5.5 incubation group was significantly higher than that of Cy5.5 group. These results suggested that the uptake ability of 8305C cells to MnO2/CDDP@PDA-Cy5.5 is better than Cy5.5.The uptake of MnO2@PDA-Cy5.5 for 24 h, the survival rate of the cells reached nearly 70% even at the concentration of 100 μg/mL. As revealed by the above result, MnO2@PDA-Cy5.5 exhibited high biocompatibility with 8305C cancer cells and Nthy-ori3-1 normal, and it can serve as a drug carrier for in-depth research.As depicted in 2/CDDP@PDA-Cy5.5. As depicted in 2/CDDP@PDA-Cy5.5 group was notably higher than that in the free CDDP group. Furthermore, at the identical concentration of CDDP, the mortality rate in the MnO2/CDDP@PDA-Cy5.5 group was significantly higher than that in the CDDP group, suggesting that MnO2/CDDP@PDA-Cy5.5 has a better ability to inhibit the growth of tumor cells. This can be attributed to the higher amount of CDDP that the drug delivery system is able to internalize in the cell.Subsequently, 8305C cells were adopted to evaluate the therapeutic effect of MnO1-C4). The apoptosis rates of 8305C cells administrated with PBS, MnO2, CDDP and MnO2/CDDP@PDA-Cy5.5 were 5.28%, 6.11%, 14.98% and 19.04%, respectively. Compared with free CDDP, MnO2/CDDP@PDA-Cy5.5 induced more apoptosis in 8305C cells. This can be attributed to the EPR effect of nanoparticles improved the targeting of nanoparticles and effectively enhanced the apoptosis of 8305C cells.The results of flow cytometry are shown in 2/CDDP@PDA-Cy5.5. With the gradual accumulation and penetration of MnO2/CDDP@PDA-Cy5.5, the fluorescence signal was detected only in the tumor area and reached its maximum intensity 4 h after injection, suggesting effective tumor accumulation by MnO2/CDDP@PDA-Cy5.5 NPs through EPR effect. Notably, fluorescence at tumor sites was still evident 8 h after injection, arising from the prolonged blood circulation of NPs and their remarkable ability to target the tumor.As depicted in in vivo imaging of MnO2/CDDP@PDA-Cy5.5 is shown in 1 imaging signal in the tumor steadily grew over time, which was peaked at 4 h. This is also confirmed by comparing the intensity of T1 signal corresponding to the tumor site and muscle 2/CDDP@PDA-Cy5.5 could be employed as an acid-responsive T1 contrast agent.The 2 groups, but after 14 days of treatment, tumor growth rate in the free CDDP group was inhibited to a certain extent. In addition, tumor volume decreased in MnO2/CDDP@PDA-Cy5.5 nude mice. Meanwhile, as depicted in 2/CDDP@PDA-Cy5.5 group exerted the optimal therapeutic effect. 2 group, the free CDDP group, and the MnO2/CDDP@PDA-Cy5.5 group reached 0.367 g, 0.227 g, 0.132 g, and 0.100 g, respectively, consistent with the growth trend of tumor volume. As revealed by the above-mentioned results, MnO2/CDDP@PDA-Cy5.5 can exert a notable therapeutic effect while outperforming free CDDP.As depicted in 2/CDDP@PDA-Cy5.5 group were organized in comparison to the free CDDP group, and no pathological alterations or necrosis were reported in the kidneys, suggesting that MnO2/CDDP@PDA-Cy5.5 mitigated the renal toxicity of CDDP. There were no significant differences in all other major organs, including the heart, liver, spleen and lungs, in the MnO2 or MnO2/CDDP@PDA-Cy5.5 groups compared with the saline group.As depicted in 2/CDDP@PDA-Cy5.5 group had tumor cell necrosis and shrinkage compared with the rest groups. Thus, the significantly improved therapeutic efficacy was shown for tumor-bearing mices administered MnO2/CDDP@PDA-Cy5.5 in comparison to those administered with CDDP at the identical CDDP doses. Based on in situ TUNEL, the apoptosis levels of tumor tissues were investigated in depth, and little green coloration was observed in the tumors of nude mice administrated with PBS and MnO2, suggesting that no cell apoptosis was identified (2/CDDP@PDA-Cy5.5 NPs. Thus, compared with free CDDP, MnO2/CDDP@PDA-Cy5.5 nanoparticles are more effective in inducing apoptosis.entified . In cont2/CDDP@PDA-Cy5.5 NPs did not induce obvious liver and kidney dysfunction (2/CDDP @ PDA-Cy5.5 NPS has good biocompatibility, and can provide passive drug delivery with low risk of off-target toxicity.As depicted in function . In a wo2+ existed in the tumor than in the other organs, suggesting that MnO2/CDDP@PDA-Cy5.5 NPs exhibited significant tumor accumulation (2+ were identified in the kidneys of the above-mentioned mice, suggesting that Pt2+ from MnO2/CDDP@PDA-Cy5.5 NPs was quickly cleared by the kidneys.The biodistribution results indicated that much more Ptmulation , probabl2 nanospheres prepared using the template method were spherical with hollow structure under the transmission electron microscope, and the average particle size reached nearly 70 nm. The stability of MnO2/CDDP@PDA-Cy5.5 NPs was notably enhanced after polydopamine modification, the particle size of the prepared MnO2/CDDP@PDA-Cy5.5 NPs was 90 nm, and the Zeta potential was 28.9 ± 3.90 mV. The results of drug release experiments showed that MnO2/CDDP @ PDA-Cy5.5 released CDDP rapidly under acidic and redox conditions, and the cumulative release reached its maximum at 8 h. As indicated by the magnetic resonance imaging results, the prepared drug delivery system was pH and redox responsive, and the degraded Mn2+ could be employed for MRI imaging. In vitro cytological research on MnO2/CDDP@PDA-Cy5.5 nanoparticles suggested that the carrier was biocompatible with 8305C cells and MnO2/CDDP@PDA-Cy5.5 could effectively kill cancer cells in vitro. Besides, a high level of uptake of MnO2/CDDP@PDA-Cy5.5 NPs by cancer cells was identified through confocal microscopy. As revealed by the in vivo zoological research on MnO2/CDDP@PDA-Cy5.5 NPs, the drug delivery system can stably deliver drugs to the tumor site, thus exerting significant inhibitory effect on the growth of the tumor. Furthermore, the drug delivery system can be employed for magnetic resonance imaging after being degraded to Mn2+ at the tumor site.The MnO2+ produced by degradation exhibited the capability of magnetic resonance imaging, such that the dual functions of tumor diagnosis and treatment can be achieved, and a certain theoretical and practical basis can be laid for the research of new preparations for tumor targeted diagnosis and treatment.The overall results suggested that the drug delivery system has pH and redox responsiveness, and can deliver drugs to tumor sites stably, and the Mn


Saccharomyces cerevisiae protein Erv14, also known as cornichon, belongs to a conserved family of cargo receptors required for the selection and ER export of transmembrane proteins. In this work, we show the importance of a phosphorylation consensus site (S134) at the C-terminus of Erv14. Mimicking phosphorylation of S134 (S134D) prevents the incorporation of Erv14 into COPII vesicles, delays cell growth, exacerbates growth of sec mutants, modifies ER structure and affects localization of several plasma membrane transporters. In contrast, the dephosphorylated mimic (S134A) had less deleterious effects, but still modifies ER structure and slows cell growth. Our results suggest that a possible cycle of phosphorylation and dephosphorylation is important for the correct functioning of Erv14.The endoplasmic reticulum (ER) is the start site of the secretory pathway, where newly synthesized secreted and membrane proteins are packaged into COPII vesicles through direct interaction with the COPII coat or aided by specific cargo receptors. Little is known about how post-translational modification events regulate packaging of cargo into COPII vesicles. The  Summary: COPII vesicle formation and trafficking of plasma membrane proteins depend on the phosphorylated state of S134 in Erv14 Saccharomyces cerevisiae begins when the GTPase Sar1 is activated by the guanine nucleotide exchange factor (GEF) Sec12, causing its incorporation into ER membranes where it recruits the inner coat layer complex, namely, the Sec24–Sec23 complex. Coat polymerization and vesicle budding occur when Sec23–Sec24 recruits a second complex, the tetramer composed of Sec13–Sec31 that forms the outer coat layer. These events lead to the hydrolysis of GTP on Sar1 and vesicle budding (S. cerevisiae Erv14 identified a cytosolic motif (IFRTL) that is required for COPII binding and ER exit of the receptor (The early secretory pathway in eukaryotic cells is a process important for the delivery of membrane and soluble proteins to various cellular destinations. Once proteins have been correctly folded and modified in the endoplasmic reticulum (ER), they are sorted from ER-resident proteins and selectively incorporated into COPII-coated vesicles that bud from the ER membrane at ER-exit sites (ERES) to be transported to the Golgi , althoug budding . The loa budding . Among t budding , as well budding . The Erv budding , most of budding . Previoureceptor . Subsequreceptor . It has receptor .S. cerevisiae, it has been reported that some COPII coat subunits are phosphorylated by the serine/threonine kinase Hrr25 (a CK1δ ortholog), allowing coat release and vesicle fusion with the Golgi  technique, which is an electrophoresis approach that allows the separation of phosphorylated and non-phosphorylated proteins using conventional SDS-PAGE procedures   at the C-terminus of Erv14 that functions as an interaction site with its cargo proteins. This acidic motif is only present in plants and fungi homologs, but not in higher organisms . To charocedures . For thie latter A. To idev14S134D B. In comted form B. For thv14S134D B. AdditiD (S134D B. To con-tag gel C. This aed (18%) C. For thlar 92%; C, suggesS134D) and S134A (Erv14S134A), respectively. When we transformed Erv14S134A or Erv14S134D into the strain BY4741Δerv14 and monitored cell growth rate, we observed a slower growth compared to the wild-type, and even slower for cells transformed with the empty vector pDR-F1  fraction, indicative of normal trafficking. In contrast, when Nha1–GFP was co-expressed with the mutant Erv14S134D, the antiporter remained in the ER (P13) fraction   and Qdr2 observing the lack of interaction between the two proteins  . For thind His−) A. The grthionine A. To conproteins A, confirproteins , and demin vivo by confocal microscopy. Erv14–GFP was found mainly in both the cortical and the perinuclear ER, with some dots that could be associated with the Golgi, as previously observed (S134A or Erv14S134D seemed to be like that observed for the wild-type receptor (S134D mutant was shifted to the ER fraction (P13), indicating its retention at the ER  with respect to the total protein  were quantified, showing that the ratio for the mutant Erv14S134D was 20% higher than that for Erv14  cells showed a clear continuous cortical ER at the TEM level  expressing HA-tagged Erv14 or the point mutants Erv14S134A or Erv14S134D, incubated with or without purified COPII proteins , GTP and an ATP regeneration system to drive vesicle budding   B. On theectively B. For thel of 4% B. These ild type . TogetheERV14, when combined with temperature-sensitive mutations involved in COPII vesicle formation, such as mutations in SEC23, SEC16 and SEC31, increased the thermosensitivity of the cells, which indicated that Erv14 was involved in the formation of the COPII vesicles  acidic motif at the C-terminus of Erv14 is required for interaction and correct targeting of its cargo proteins   C. Particd SEC13) , it mighhttps://www.deepmind.com; Based on the cornichon molecular model  and the sec mutants, both Erv14S134D and Erv14S134A cause deformations in the ER and worsen cell growth  to disrupt the phosphorylation domain without modifying S134. This modification led to the disappearance of the upper band  at the C-terminus for the controlled transport of cargoes. Owing to the neutral pH of the ER lumen, the acidic amino acids might establish an electrostatic interaction with cargo proteins, allowing their loading into the COPII vesicles, where S134 would not play any role as it would be unphosphorylated. Upon arrival at the Golgi, as it has been previously described  to YNB plates and growth at 23°C, 28°C or 30°C, and cell growth was recorded for 3 days. To monitor growth rate, yeast cells were preinoculated in selective medium and the next day an optical density at 600 nm (OD600) of 0.01 was inoculated in YNB medium and growth was monitored every 2 h in a spectrophotometer . Yeast cells were transformed using a standard lithium acetate transformation protocol  and employing the specific primers described in S134AERV14 and S134DERV14 were cloned into the pDONOR207 vector of the Gateway system following manufacturer's instructions . The entry clones thus generated were confirmed by restriction analysis with EcoRI and BgllI  and by sequencing. The LR clonase reaction  was used to transfer the genes S134A and ERV14S134DERV14, ERV14 into the multicopy plasmid pAG425-3HA (Addgene plasmid #14250), to generate the HA-tagged fusions pERV14-3HA, pERV14S134A-3HA and pERV14S134D-3HA. To complement ERV14 function in the BYT45erv14Δ, BY4741pdr12Δerv14Δ and BY4741qdr2Δerv14Δ mutant strains, the genes ERV14, S134AERV14 and S134DERV14 were cloned into the pDR-F1_GW (Addgene plasmid #36026) by an LR clonase reaction  generating the constructs pDR-F1-ERV14, pDR-F1-ERV14S134A and pDR-F1-ERV14S134D. For the mbSUS assay constructs, the genes S134AERV14 ERV14 and S134DERV14 were transferred into the pXN32_GW (Nub clones) vector by an LR clonase reaction , generating the pNub-ERV14, pNub-ERV14S134A and pNub-ERV14S134D. The pQDR2-Cub (Cub clone) construction used in this work was obtained from a previous work  and by sequencing, using the oligonucleotides described in the To generate the point mutations S134A and S134D, the ORF of ous work . To geneMATa ura3, leu2, lexA::LacZ::trp1 lexA::HIS3 lexA::ADE2) and THY.AP5  yeast strains were transformed with the pQdr2-Cub (Cub clones) and pNub-Erv14S134A or pNub-Erv14S134D (Nub clones) constructions, respectively. Cells were allowed to mate on YPD medium for 24 h at 30°C, and then, diploid cells were selected on SC medium. For testing interactions, serial dilutions of cells were applied on SC medium supplemented with 5 µM to 500 µM methionine, and cell growth was recorded for 3 days. The β-galactosidase activity of the lacZ  and cell pellets resuspended in (25 µl) of YNB medium. 5 µl of resuspended cells were placed on a slide and were observed under an inverted multiphotonic confocal microscope (Olympus FV1000) equipped with a 60×1.3 NA oil immersion objective. GFP fluorescence was visualized by excitation with a multi-line Argon laser at 488 nm and spectral detector was set between 515/30 nm for emission. Images were processed with ImageJ  of the cultures was harvested, centrifuged at 2795  h ImageJ .600 of 0.1 in 50 ml of YPD medium the following morning, and then grown to approximately an OD600 of 1.0 and harvested by centrifugation in an Sorvall RC 6 Plus centrifuge  at room temperature. The cell pellet was resuspended in buffer , incubated for 10 min at room temperature and centrifuged as before. The cells were resuspended and converted into spheroplasts by digestion with (+)-lyticase buffer . The cells were incubated at room temperature until the OD600 of a 1:100 dilution in H2O was less than 80% of the initial value and then spheroplasted cells were diluted into ice-cold (−)-lyticase buffer  followed by centrifugation at 2795 g  for 5 min at 4°C. Spheroplasts were resuspend in B88 buffer , aliquoted, frozen in liquid nitrogen and stored at −80°C. Spheroplasts were used as a membrane source in the vesicle budding assay. Isolation of ER- and Golgi-enriched fractions followed a similar procedure. After converting into spheroplasts, pelleted cells were resuspended in 2.5 ml JR lysis buffer  and lysed with six strokes of a Dounce homogenizer and centrifuged in an SS-34 rotor at 447 g at 4°C for 5 min, to pellet nuclei and unbroken cells. The supernatant was recovered and centrifuged at 24,104 g for 10 min at 4°C in an Eppendorf 5417R centrifuge to generate the ER-enriched P13 pellet fraction. The supernatant was recovered and centrifuged at 100,000 g for 15 min at 4°C in an Optima TL micro-ultracentrifuge  to obtain the Golgi-enriched p100 pellet fraction. The P13 and P100 pellets were resuspended in 100 µl JR lysis buffer and diluted 1:1 with 5× sample buffer , boiled, and separated on 12% SDS-PAGE gels. Immunoblotting was conducted with polyclonal antibodies against Yet3  or (Erv14S134D) in BY4742Δerv14 cells and incubated in the presence or absence of purified COPII proteins , GTP and an 10× ATP regeneration system, and incubated at 25°C for 30 min, for reconstitution of vesicle formation  to collect the membranes. Membrane pellets were resuspended in 25 µl of sample buffer 5×, and 8 or 10 µl aliquots were loaded on 14% polyacrylamide gels and immunoblotted (see below) for Sec61, Erv41, Coy1 or with anti-HA for Erv14–3HA (WT) or mutants Erv14S134D–3HA, Erv14S134A–3HA detection. To estimate packaging efficiency, the intensity of the bands was quantified using GeneTools image analysis software (Syngene), where (T) represents 10% of the reaction and was used to compare that from the vesicles obtained from the different Erv14 isoforms.The COPII vesicle budding assay was performed as previously described . Washed ormation . A pool For sample preparation, three independent microsomes replicates (10 µg per replicate) were run on an SDS-PAGE. Protein spots from the gel that corresponded to the bands recognized by the HA antibody were sent to the Proteomics Facility at the Institut de Recherches Cliniques de Montréal, Canada. The samples were analyzed using Mascot software . Mascot was set up to search the Uniprot_S_cerevisiae_txid_4932 database. Scaffold  was used to validate LC-MS/MS-based peptide and protein identifications. Scaffold parameters were set to a minimum of two peptides per protein and accepted if they could be stablished with minimum probabilities of 99% at the protein level by the Peptide Prophet algorithm.g for 20 min at 4°C using an F2402 rotor in a GS15R table-top centrifuge (Eppendorf). Pellets were air dried, resuspended with 4× SDS-PAGE sample buffer , and heated at 95°C for 5 min before loading onto 12% (w/v) linear acrylamide mini-gels. For vesicle budding assay samples, pellets were resuspended in sample buffer 4×, boiled at 95°C for 5 min, and resolved onto 14% (w/v) SDS-PAGE gels. After protein separation, gels were either fixed and stained with Coomassie Brilliant Blue R 250 or electrophoretically transferred onto nitrocellulose membranes (Millipore) by standard methods. Following transfer, proteins were stained with Ponceau S protein stain (0.1% w/v in 1% v/v acetic acid for 30 s) to check for equal loading/transfer of proteins. Membranes were then blocked with TBS  containing 0.02% (w/v) Na-azide and 5% (w/v) fat-free milk powder  for 2 h at room temperature. Blocked membranes were incubated overnight with the appropriate primary antibodies, anti-HA , anti-phospho-serine , anti-GFP , anti-Erv41 and anti-Och1  ethanol:acetone and incubated overnight at −30°C as described . Samples2, 100 µl of 10% SDS, 10 µl TEMED, 25 µl of 10% APS and 4.5 ml of distilled water] was used. The stacking gel was prepared with 600 µl of 30% acrylamide-bis-acrylamide , 1 ml of 0.5 M Tris-HCl pH 6.8, 40 µl of 10% SDS, 4 µl TEMED, 20 µl of 10% ammonium persulfate and 2.34 ml of distilled water. The gel was run at 30 mA for 6 h at 4°C. After electrophoresis, the resolving gel was washed three times in 100 ml of transfer buffer  and then washed once for 20 min in transfer buffer without EDTA. Protein was blotted onto nitrocellulose membrane at 30 V (at constant voltage) for 8 h at 4°C. Membranes were blocked using 5% nonfat milk and probed with 1:10,000 dilution of anti-HA .Phos-Tag SDS-PAGE protein separation was performed according to manufacturer's specifications . The Phos-tag gel was prepared with 10% acrylamide and 50 µM Phos-tag as described below. For the resolving gel, 2.67 ml of solution [30% acrylamide-bis, 2.5 ml of 1.5 M Tris-HCl pH 8.8, 100 µl of 5 mM Phos-tag , 100 µl of 10 mM ZnCl600 0.8). Cells were centrifuged at 1500 g for 5 min in an F2402 rotor in a GS15R table-top centrifuge (Beckman) and resuspended in water to wash the cells. The pellet was fixed in ice-cold fixing cacodylate buffer  for 2 h at 4°C and centrifuged as above. Cells were resuspended in cacodylate buffer and incubated overnight at 4°C. Cells were pelleted as above and washed three times in cacodylate buffer; then, cells were resuspended in 2% KMnO4 for 1 h at room temperature. After secondary fixing, cells were resuspended in 1 ml of freshly prepared 2% uranyl acetate for 1 h at room temperature and subsequently washed in water. Dehydration of cells was followed by clearing with propylene oxide and infiltration with a combination of 100% propylene oxide and 100% Spurr low-viscosity resin . Finally, samples were embedded in 100% resin and kept in a vacuum oven at 45°C for 12 h, then shifted to 68°C for 3 days. For TEM examination, thin sections of grey-silver color interference (60–70 nm) were cut and mounted onto 300-mesh copper grids. The mounted sections were stained with alkaline lead citrate, washed gently with distilled water and allowed to dry for 1 h. Dried sections were observed using a CARL ZEISS Libra 120  transmission electron microscope, at a voltage of 80 kV, coupled to a Gatan Ultrascan 1000 CCD (2000×2000 pixels)  to record the images.For transmission electron microscopy, samples were prepared as described previously . Yeast cClick here for additional data file.10.1242/joces.260527_sup1Supplementary informationClick here for additional data file.


NPM1 is a phosphoprotein highly abundant in the nucleolus. However, additional nuclear functions have been attributed to NPM1, probably through interaction with other nuclear factors. DOT1L is one interaction partner of NPM1 that catalyzes methylation of histone H3 at lysine 79 (H3K79). DOT1L, playing functional roles in several biological processes, is known for its capability to organize and regulate chromatin. For example, DOT1L modulates DNA repeats expression within peri-nucleolar heterochromatin. NPM1 also affects peri-nucleolar heterochromatin spatial organization. However, it is unclear as of yet whether NPM1 and DOT1L functionally synergize to preserve nucleoli organization and genome stability, and generally, which molecular mechanisms would be involved.Ezh2; (iv) NPM1 and DOT1L modulate DNA repeats expression and peri-nucleolar heterochromatin organization via epigenetic mechanisms dependent on H3K27me3.We characterized the nuclear function of NPM1 on peri-nucleolar heterochromatin organization. We show that (i) monomeric NPM1 interacts preferentially with DOT1L in the nucleus; (ii) NPM1 acts in concert with DOT1L to maintain each other’s protein homeostasis; (iii) NPM1 depletion results in H3K79me2 upregulation and differential enrichment at chromatin binding genes including Our findings give insights into molecular mechanisms employed by NPM1 and DOT1L to regulate heterochromatin activity and structural organization around the nucleoli and shed light on one aspect of the complex role of both proteins in chromatin dynamics.The online version contains supplementary material available at 10.1186/s13072-023-00511-9. NPM1 is a highly abundant phospho-protein in the granular region of the nucleolus, it contains a nuclear localization signal (NLS) and can shuttle between the nucleus and cytosol  is a prominent interaction partner of NPM1 . DOT1L iIn addition to controlling several aspects of transcriptional regulation, DOT1L participates in different cellular processes such as cell proliferation , embryonLike NPM1, DOT1L has been implicated in the pathogenesis of AML, bearing the MLL-AF9 (MLLT3)/ AF10 (MLLT10) fusion proteins. Here, through its association with the transcription factors AF9 and AF10, DOT1L is recruited to MLL target genes, which results in their aberrant methylation at H3K79 and the establishment of the malignant phenotype , 47.Despite an increasing, but so far limited understanding of individual DOT1L and NPM1 functions, our knowledge of the implications of their cooperation and crosstalk is even less complete. A few insights come from studies in NPM1c + AML , 27, in Other functional associations between NPM1 and DOT1L can be anticipated. On one hand, NPM1 expression maintains the nucleolar shape and peri-nucleolar heterochromatin organization . This cIn this work, we addressed a potential synergy between mouse NPM1 and DOT1L, with a specific focus on heterochromatin organization around nucleoli in the neuroblastoma N2a cell line. We show that monomeric NPM1 interacts with DOT1L in the nucleus at the chromatin level. Extending on individual, yet synergistic functions, we identified a novel regulatory feedback loop that involves both NPM1 and DOT1L. NPM1 and DOT1L modulate the epigenetic landscape and contribute to the maintenance of DNA repeats silencing within peri-nucleolar heterochromatin, whereas “DNA repeats” refer to both tandem repeats (DNA satellites) and interspersed repeats . Mechanistically, NPM1 and DOT1L contribute to the organization of heterochromatin around nucleoli through a crosstalk with H3K27me3. This study of NPM1 and DOT1L cooperation is of crucial relevance to understand the multifaceted ways of action of both proteins. Our finding of a synergistic role of NPM1 and DOT1L in maintaining heterochromatin functions around the nucleoli, and consequently in genome stability, provides a possible molecular mechanisms to explain malignant phenotypes of some cancer cells (for example NPM1c + types of leukemia), and thus has also important clinical implications.Previous data showed co-immunoprecipitation (co-IP) of NPM1 with the methyltransferase DOT1L in human HEK293T cells , but furThe intracellular localization of NPM1 is regulated by its RNA binding activity and oligomerization . RNA binTo test whether NPM1 would have regulatory effects on DOT1L activity impacting H3K79 methylation similar to human cells , we reduDot1l accounted for the increased levels of H3K79me2 upon NPM1 KD. By employing RT-qPCR we identified an increased expression of Dot1l in the NPM1 KD condition  with an H3K79me2 specific antibody identified the genomic regions that enriched differentially in H3K79me2 upon NPM1 KD. Two biological replicates were used, and immunoblots verified decreased NPM1 and increased H3K79me2 levels, respectively . Of furEzh2 and Ctcf, alongside Dot1l and an almost significant enrichment at Npm1 ) alone had strong and variable batch effects on DNA repeats transcription  functions as a coactivator of RNA polymerase II-dependent transcription to promote the expression of genes involved in tumorigenesis . Given tNPM1/DOT1L binding is also influenced by RNA, because RNase treatment increased the association of the two partners. RNA as critical regulator of NPM1/DOT1L interaction is further supported by our finding of a preferential interaction of DOT1L with monomeric NPM1. RNA prevents NPM1 oligomerization and translocation into the nucleoli, which might result in increased amounts of NPM1 in the monomeric form that is consequently available for interaction with DOT1L .Dot1l transcription, whereas DOT1L is recruited to the Npm1 promoter and its enzymatic activity is required to activate NPM1 expression. Our study did not unravel as of yet whether NPM1 and DOT1L proteins act independently or as a complex to target their own genes. However, this assumption is not unlikely, as a direct role of NPM1 as a transcriptional regulator has been recently shown in NPM1c +AML [A further crucial finding of our study is the existence of a regulatory feedback loop that involves both NPM1 and DOT1L. In this context, NPM1 represses M1c +AML .The significance of this regulatory mechanism in which NPM1 and DOT1L influence the expression of their own genes is underlined by the importance of NPM1 and DOT1L homeostasis for cell viability and development. NPM1 overexpression is considered a prognostic marker for the recurrence and progression of various hematological malignancies  and highEzh2 upon NPM1 KD  the different efficiency of NPM1 KD in each biological replicate, (ii) batch effects across experimental conditions, and (iii) the heterogeneous nature of the N2a cells might contribute as well. Given the high variability in these experiments, we cannot conclude that the increased global levels of H3K27me3 depend alone on increased EZH2 levels, although its expression is altered upon NPM1 KD. It is still possible that the increase of H3K27me3 at peri-nucleolar DNA repeats occurs independently of changes in EZH2 protein levels. One alternative scenario could be that upon NPM1 KD, DOT1L recruitment at peri-nucleolar heterochromatin affects chromatin accessibility, favoring binding of EZH2, and increasing H3K27me3 at DNA repeats, independent from a direct transcriptional control of Ezh2. In this regard, we have previously demonstrated that in neural progenitor cells, DOT1L inactivation leads to reduced H3K27me3 levels at selected target genes, without impacting Ezh2 expression, but by altering PRC2 recruitment [In the presence of limiting amounts of NPM1, H3K79me2 enrichment at the promoter of chromatin binding genes correlates with variable changes in their expression. Although we did not detect strong changes in the transcription of DOT1L target genes upon NPM1 KD, these subtle changes are expected in agreement with DOT1L being a modulator of transcription rather than a transcriptional activator , 21. Seeruitment .Our ChIP-seq analysis unraveled more regions with decreased than increased levels of H3K79me2  . These nIn addition, we detected changes in peri-nucleolar heterochromatin activity that is, the enhanced silencing of repetitive regions in NPM1-depleted cells compared to controls. Heterochromatin surrounding the nucleoli consists of repetitive major satellite (pericentric) and minor satellite (centric) DNA sequences and of tandemly repeated ribosomal RNA (rRNA) genes . CorrectOur findings of a non-canonical role of DOT1L in DNA repeats repression are supported by the recent discoveries of Zhao et al. , who shoOur findings of the critical role of the NPM1 and DOT1L in peri-nucleolar heterochromatin functions and organization in N2a cells sheds new light on the versatile roles of NPM1 and DOT1L in chromatin regulation. Moreover, our data advance our understanding of how NPM1 and DOT1L might be recruited to specific target regions, i.e. through interaction with specific protein partners.Despite much more research is needed to gain full insights into how NPM1 cooperates with DOT1L to impact nucleoli activity, and into the full functional significance of the NPM1/DOT1L protein complex, our findings provide a valuable resource for future studies on how the epigenome contributes to the organization of the nucleolus.Mouse neuroblastoma cell line, Neuro-2a (N2a) was cultured and maintained at 37 °C, 95% relative humidity and 5% CO2 in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 1% non-essential amino acids , 1% L-glutamine, and 1% penicillin, streptomycin, and neomycin . Cells were seeded in 24 wells plates on coverslips either for proximity ligation assay (PLA) immunofluorescence and DNA-FISH, or in 6 wells plates for biochemistry and ChIP/RT-qPCR. Cells were transfected with Lipofectamine LTX according to the manufacturer´s instructions (ThermoScientific). For NPM1 knockdown (NPM1 KD) and DOT1L overexpression (DOT1L OE) experiments, N2a cells were selected with 9.3 µg/ml Puromycin starting 24 h after transfection and for the remaining time until fixation or collection. Where indicated 10 μM EPZ5676  in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was added to N2a cells for 72 h and refreshed every second day. DMSO was used as control treatment. The following plasmids were used for transfection: pLKO.1-shCTR , pLKO.1-shNPM1 , pLenti-CMV-HA-2A-GFP (Genscript), pLenti-CMV-DOT1L-FLAG-HA-2A-GFP (Genscript) and pVB-DOT1L-FLAG (Vector Builder).For total cell lysate preparation frozen cell pellets were lysed in Lysis Buffer  supplemented with complete Protease Inhibitor cocktail (Roche-Diagnostics). Cells in Lysis Buffer were kept on ice for 30 min and chromatin disruption was done by sonication for 4 min in AFA 130 μl tubes (Covaris) using the Covaris E220. Cell debris was removed by centrifugation at 14.000 rpm 4 °C 5 min. The supernatant was collected and protein concentrations were quantified photometrically with Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad). 5–15 μg of cell lysates were loaded on 4–20% gradient SDS-PAGE (Bio-Rad). Proteins were transferred to PVDF membranes or to nitrocellulose membranes for Ponceau staining using the Trans-blot Turbo Transfer System (Bio-Rad) following the manufacturer’s instructions. Upon transfer, membranes were further processed for immunoblot following standard procedures.For co-IP experiments, the cell pellet was resuspended in co-IP Buffer 1  and cell lysate was left 30 min on ice. Debris was removed by centrifugation at 14.000 rpm 4 °C 5 min. The cell lysate was incubated with 2 μg of the indicated antibody bound to protein G dynabeads  overnight. The immunocomplexes were then washed with co-IP Buffer 1 three times and separated by SDS-PAGE. Immunoblotting was performed following standard procedures.7 cells) was resuspended in co-IP Buffer 2  and the cell lysate was left 30 min on ice. Debris was removed by centrifugation at 14.000 rpm 4 °C 5 min. The cell lysate was equilibrated to 150 mM NaCl by adding drop by drop co-IP Buffer 2 without NaCl. 2 μg of the indicated antibody bound to protein G dynabeads  was added overnight and cell immunocomplexes were further treated as described before.For endogenous NPM1/DOT1L co-IP, cell pellet (4*10For NPM1 cross-linking with EGS (ethylene glycol bis(succinimidyl succinate), ThermoScientific) N2a cells were transfected using Lipofectamine 2000 and allowed to express DOT1L-HA-FLAG for 72 h. The cells were then lysed in co-IP Buffer 1 and incubated with either DMSO or 0.5 mM EGS for 30 min at 25 °C. Crosslinking was quenched with the addition of 0.025 mM Tris–HCl pH 7.5 for 15 min at 25 °C. DOT1L immunoprecipitation was performed using HA antibody as described above.For nucleic acid digestion, the cell extract in co-IP Buffer 1 was treated with 20 μg of RNase (Promega) or 5U of DNase (Promega) for 30 min before performing the co-IP as described above.Cell fractionation was performed by resuspending cell pellets in 500 μl Lysis Buffer  followed by incubation on ice for 20 min. Nuclei were centrifuged at 2.000 rpm for 10 min at 4 °C and the supernatant was collected as the cytoplasmic fraction. Nuclei were then resuspended in 100 μl Nuclei Lysis Buffer  and incubated on ice for 5 min. Nuclei were pelleted at 15.000 rpm for 5 min at 4 °C and the supernatant was removed as the nucleoplasm fraction. The volume of the nucleoplasm fraction was adjusted to 250 μl with co-IP Buffer  with Protease Inhibitor cocktail. The chromatin pellet was then resuspended in 250 μl co-IP Buffer  with Protease Inhibitor cocktail and sonicated with the use of Bioruptor (Diagenode) with the following setting: cycle number: 9 + 9 Time ON: 30 s Time OFF: 45 s. The chromatin and nucleoplasm fraction were the used for co-IP as described before.The following antibodies were used for co-IP or immunoblots: anti-DOT1L , anti-DOT1L , anti-HA-tag , anti-H3 , anti-NPM1 , anti-GAPDH , anti-Actin  anti-H3K79me2 , anti-H3K27me3 , anti-H3K9me2 , anti-H3K9ac . Densitometric analyses were done with ImageJ software.Cells were seeded in multi-well plates and transfected as described before. Medium was removed and cells were washed 3 times with PBS. Fixation was performed with 4% paraformaldehyde  in PBS for 15 min at RT. After PFA removal, cells were washed 3 times in PBS. Cells were permeabilized with 0.1% Triton-X100/PBS for 15 min and blocked in 10% horse serum/PBS 1 h. Primary antibody incubation was done overnight at 4 °C in blocking solution. Cells were washed 3 times in PBS and incubated with fluorescent secondary antibodies in blocking solution at room temperature for 2 h. After washing 3 times with PBS, cells were incubated for 5 min with DAPI  and washed 3 times in PBS. Coverslips were mounted on glass slides with fluorescent mounting medium . The following first and secondary antibodies were used: LAMINB1 , FIBRILLARIN-Alexa488  H3K79me2 , NPM1 , GFP , donkey-anti-goat-Alexa488  or -Cy3 , donkey-anti-rabbit-Alexa488  or -Alexa594 , donkey-anti-chicken-Alexa488 , donkey-anti-mouse-Alexa488  or -Alexa594 , donkey-anti-rat-Alexa488  or -Alexa594  and donkey-anti-rat-AMCA .The immune-FISH cells were initially treated as for the immunofluorescence experiment with the following changes: PFA-fixed cells were permeabilized with 0.2% TritonX100 in PBS, 10 min, then wash with 1 × PBS, 3 times, 5 min; primary and secondary antibody were incubated in PBG 1 × blocking buffer  and 0,5% BSA in PBS). After washing out the secondary antibody with 1 × PBG, twice, 5 min and with 1 × PBS, twice, 5 min, cells were fixed again with PFA 4% (Life Technologies) with triton 0.1%, 10 min RT, followed by incubation with glycine 10 mM in H2O, 30 min, RT. Cells were then washed with 1 × PBS, 3 times, 5 min. For each coverslip 20 μl of hybridization mixture ) containing 0.5 μM of each DNA probe for major satellite repeats  nucleolar stained structures and the mSat foci were quantified using ImageJ with the ‘Analyze particle’ option. Particle size was set at 0.2-3000µmPLA was performed with the Duolink starter kit reagents  according to manufacturer's instructions using antibodies against FLAG  and NPM1 . After completion of the PLA protocol, we proceeded with incubation of a third antibody produced in rabbit to detect cellular compartments such as nucleolus with FBL  or nuclear membrane with LMNB1  following the procedure for immunofluorescence. Images were obtained with a Leica SP8 confocal microscope and processed with the LASX software.N2a cells were transfected with shNPM1 (NPM1 KD) or shControl plasmid (CTR) and were grown on 6 wells plates (~ 6 Mio cells) for 72 h. Two independent biological replicates per condition was used. Each biological replicate corresponds to a different passage of N2a cells and for each biological replicate transfection was performed in a separate batch. N2a cells were fixed with freshly prepared room temperature 1% PFA (Life Technologies) for 5 min at room temperature. Fixation was stopped with 125 mM glycine, and fixed cells were washed 2 times with ice-cold PBS. Cells were collected in ice-cold PBS and centrifuged 5 min at 500 rpm. Lysis Buffer  was added to the pellet and incubated on ice for 5 min. Shearing was done using Bioruptor with the settings: 3 × 10 min 30 s pulse, 30 s pause and chromatin was centrifuged for 5 min at 13.000 rpm and the chromatin samples were saved. Chromatin samples were precleared with protein G dynabeads (Invitrogen). Genomic DNA regions of interest were isolated using 4 μg of H3K79me2 antibody . Complexes were washed, eluted from the beads with SDS Buffer , and subjected to RNase and Proteinase K treatment. Crosslink was reversed by incubation overnight at 65 °C, and ChIP DNA was purified by Qiagen MinElute kit according to the manufacturer’s instructions. Quantification was performed by using PicoDrop and the PicoGreen quantification kit (Lumiprobe). Libraries were prepared using the NEBNext Ultra II DNA library preparation kit for Illumina (Biolabs) and sequenced using HiSeq 3000 (Illumina) . Galaxy platform was used for quality control, mapping, peak calling and differential enrichment analyses . High quN2a cells were transfected with pVB-DOT1L-FLAG (Vector Builder) or pLKO.1-shCTR , pLKO.1-shNPM1  as indicated and selected as described before. Chromatin was prepared as described for the H3K79me2 ChIP-seq, except for DOT1L ChIP by FLAG antibody. In this case, cells were treated with 1.5 mM EGS (ethylene glycol bis(succinimidyl succinate) for 30 min at RT before fixation with 1% PFA (Life Technologies) for 15 min at RT. The following antibody were used for the IP: H3K27me3 ; H3K79me2 ; FLAG . Primers used for ChIP-qPCR are listed in Additional file Total RNA was extracted using the RNeasy kit (Qiagen). Reverse transcription was performed using the RevertAid RT Reverse Transcription kit (ThermoScientific) following the user´s manual instructions. Primers used for RT-qPCR are listed in Additional file 6 cells) were harvested after transfection and washed with PBS. Cells were resuspended in 0.5 ml of PBS and fixed by adding the cell suspensions drop by drop to 4.5 ml 70% ethanol and stored overnight at 4 °C. The fixed cells were centrifuged at 200 g 5 min RT and washed with PBS three times. Cells were then resuspended in 1 ml of freshly prepared PI/Triton X-100 staining solution with RNase A ) and incubated for 3 h at RT before being analyzed by flow cytometry.N2a cells  Immunoblot analysis DOT1L/NPM1 interaction in N2a cell extract using either DOT1L antibody (DOT1L-IP) or NPM1 antibody (NPM1-IP). The asterisk indicates the position of endogenous DOT1L. B) top) single fluorescence channels corresponding to Fig. 1B; bottom) controls for proximity ligation assay (in situ PLA), showing stainings without both primary antibodies (left upper two panels), omitting one primary antibody at a time  or in presence of both primary antibodies (lower right two panels). In all cases, HA was used as DOT1L transfection control. . C) top) immunoblot analysis of DOT1L co-IP in control conditions or after EGS protein crosslinking using NPM1 antibody.We were not able to detect DOT1L in EGS conditions, probably due to the large size of protein complexes formed that are not able to enter the gel. The lower arrow indicates monomeric NPM1. The upper arrow indicates oligomeric NPM1 and in complex with additional proteins. Dash lines indicate the 55kDa (monomeric NPM1) and 130kDa (oligomeric NPM1) protein marker bands; bottom) the same membrane as in the top panel, left side, but probing was done against DOT1L using HA antibody. Figure S2 A) Picture of N2a cells growing in DMEM medium 3 days after NPM1 KD. Cells depleted of NPM1 (KD) consume less culture medium (pink medium) compared to control cells (yellow medium). B) Immunofluorescence image showing an example of N2a nuclei after 3 days of NPM1 knockdown (NPM1 KD) using GFP antibody to mark the transfected cells (red) and activated caspase 3 antibody  to mark cells undergoing apoptosis. Arrow indicates a cell undergoing nuclear fragmentation (NF) as shown by the lack of DAPI staining. . Images were taken using an AxioImager M2 fluorescence microscope with a 40x objective. C) Data point plot showing the percentage of transfected cells (GFP positive) expressing aCASP3 or undergoing nuclear fragmentation (NF) after 3 days of NPM1 knockdown. Statistical analysis was performed on n=3 biological replicates per condition on a total of n=176 (CTR), n=52 (KD) cells using an unpaired two tailed t-test; *p < 0.05, **p < 0.01. Error bars represent S.D. D) Flow cytometer analysis evaluating the cell cycle progression of control (CTR) and after 3 days of NPM1 knockdown (NPM1 KD) in N2a cells (left two panels). Stacked bar graphs showing the percentage of cells in different phases of the cell cycle in both conditions  (right panel). Statistical analysis was performed on n=3 biological replicates using a two-way ANOVA with Sidak’s post-hoc test. *p < 0.05, **p < 0.01. No significant differences were observed. Figure S3. A) Immunoblot of the two biological replicates used for the H3K79me2 ChIP-seq showing reduction after 3 days of NPM1 (KD) and concomitant increase of H3K79me2. GAPDH immunoblot was used as the normalization control.. B) Venn diagram depicting the intersection of H3K79me2 peaks in control (CTR) and after 3 days of NPM1 knockdown (NPM1 KD) in N2a cells.. C) Heatmap of H3K79me2 enrichment at the indicated genes (top) in control (CTR), after 3 days of NPM1 knockdown (NPM1 KD), and NPM1 KD/CTR conditions clustered into regions found 1 Kb up-/down-stream of scaled regions. Data is normalized by sequencing depth and input control . The metaprofiles show the log2(Fold Change) (LFC) of each gene. The scale corresponds to the log2(ChIP/Input) for control and NPM1 KD and to log2(ChIP NPM1 KD/ChIP CTR) for NPM1 KD/CTR.. D), E) Modified Integrative Genome Viewer (IGV) snapshot depicting the normalized H3K79me2 levels (Log2(H3K79me2 ChIP/Input)) at Ezh1 and Kdm3a genes in control (dark blue) or upon 3 days of NPM1 KD (light blue) cells, and the H3K79me2 signal ratio between NPM1 depletion and control . The exact positions of the genes within the mouse mm10 genome and of the primers used for ChIP-qPCR are indicated at the bottom. Coverage is auto-scaled to fit the window. Figure S4. A) Modified Integrative Genome Viewer (IGV) snapshot depicting the normalized H3K79me2 levels (Log2(H3K79me2 ChIP/Input)) at Ctcf gene in control (dark blue) or upon 3 days of NPM1 KD (light blue), and the H3K79me2 signal ratio between NPM1 depletion and control . The exact positions of the genes within the mm10 genome and of the primers used for ChIP-qPCR are indicated at the bottom. Coverage is auto-scaled to fit the window. B) Individual data points plot showing the fold change (NPM1 KD/CTR) expression of the indicated genes calculated after RT-qPCR upon 3 days of NPM1 KD in N2a cells. Gapdh was used as reference gene for normalization. Statistical analysis was performed on n=9 biological replicates using an unpaired two tailed Student's t-test; *p < 0.05, **p < 0.01. Outliers were identified using a Grubbs method. Error bars represent S.D. C) Line chart showing the dose dependent expression of Ezh1 (red lines) and Ezh2 (blue lines) to high Dot1l levels, or moderately increased Dot1l levels in black lines upon 3 days of NPM1 KD compared to controls, calculated after RT-qPCR. D) Immunoblot analysis of the levels of H3K79me2 upon 3 days of NPM1 KD in N2a cells. Ponceau staining was used for total protein normalization. E) Quantification of the signal intensities of the immunoblots shown in D, and in Fig. 4B, using Fiji (ImageJ). Protein levels were first normalized to the corresponding Ponceau signal and then presented as a ratio over the control values. Statistical analysis was performed on n=3 biological samples using an unpaired two tailed t-test. *p < 0.05, **p < 0.01. Error bars represent S.D. F) Individual data points plot showing the fold change expression (FC) of the indicated genes after RT-qPCR upon 3 days of NPM1 KD (blue) or DOT1L OE (red) in N2a cells. Gapdh was used as reference gene for normalization. Statistical analysis was performed on n=4 biological replicates using an unpaired one tailed (Npm1 and Dot1l) and two tailed t-test . *p < 0.05, **p < 0.01. Outliers were identified using a Grubbs method. Error bars represent S.D. Figure S5. A) Immunoblot analysis of the levels of the H3K9ac, H3K9me2, H3K79me2 and NPM1 upon 3 days of NPM1 KD in N2a cells. Ponceau staining was used for total protein normalization. B) Quantification of the signal intensities of H3K9me2 and H3K9ac shown in A was done using Fiji (ImageJ). Protein levels were first normalized to the corresponding Ponceau signal and then presented as a ratio over the control values. Statistical analysis was performed on n=4 biological samples using an unpaired two tailed t-test. *p < 0.05, **p < 0.01. Error bars represent S.D. C) Individual data points plot showing the fold change enrichment over control of H3K79me2 calculated after ChIP-qPCR upon 3 days of NPM1 KD (KD/CTR). Abbreviations are: gSAT (gamma satellites), mSAT (major satellites). Statistical analysis was performed on n=4 biological replicates using an unpaired two tailed t-test. *p < 0.05, **p < 0.01. Error bars represent S.D. Figure S6 A) Confocal immunofluorescence of N2a nuclei after 3 days of NPM1 KD and treatment with either EPZ or DMSO as control using FBL (FIBRILLARIN) antibody to highlight nucleolar shape. Note that CTR cells have larger and fewer nucleolar structures compared to NPM1 KD. EPZ treatment reverts only partially this phenotype, although the change is not statistically significant . B) Quantification of the mean number (left) and area (right) of FBL (FIBRILLARIN) nucleolar stained structures shown in A. Statistical analysis was performed on n=2 biological replicates andthe following total number of nuclei: n=41 (CTR), n=35 (KD), n=17 (KD+EPZ) and n=35 (CTR+EPZ) to measure the number of nucleoli per nuclei , and n=24 (CTR), n=32 (KD), n=24 (KD+EPZ) and n=37 (CTR+EPZ) to measure the area per each condition using a two-way ANOVA with Sidak’s posthoc test. *p < 0.05, **p < 0.01. Error bars represent S.D. C) Confocal immunofluorescence of N2a nuclei after 3 days of NPM1 KD and treatment with either EPZ or DMSO as control using H3K79me2 antibody. Note that KD cells have increased H3K79me2 levels. EPZ treatment decreases H3K79me2 staining intensity. . Figure S7 A) and B) Modified Integrative Genome Viewer (IGV) snapshots depicting the normalized H3K79me2 levels (Log2(H3K79me2 ChIP/Input)) at Npm1 and Dot1l genes in control (dark blue) or upon 3 days of NPM1 KD (light blue), and the H3K79me2 signal ratio between NPM1 depletion and control . The exact positions of the genes within the mm10 genome and of the primers used for ChIP-qPCR are indicated at the bottom. Coverage is auto-scaled to fit the window. C) Representative immunoblot analysis of the levels of NPM1 and H3K79me2 upon 3 days of NPM1 KD in N2a cells. Ponceau staining was used for total protein normalization. D) Quantification of the signal intensities shown in C was done using Fiji (ImageJ). Protein levels were first normalized to the corresponding Ponceau signal and then presented as a ratio over the control values. Statistical analysis was performed on n=5 biological samples using an unpaired two tailed t-test. *p < 0.05, **p < 0.01. Error bars represent S.D. E) Confocal immunofluorescence of N2a cells showing NPM1 distribution in control transfected cells (GFP) or cells after 3 days of overexpressing DOT1L (DOT1L OE). . F) Confocal immunofluorescence of N2a cells showing NPM1 localization in DMSO treated cells (CTR) or cells treated for 48 hours with the DOT1L inhibitor EPZ. . Figure S8 A) Immunoblot analysis confirming the overexpression of DOT1L (FLAG) after 3 days in N2a cells. Ponceau was used for total protein normalization. Note that the levels of H3K79me2 are also increasing.Additional file 2: Table S1. List of genes with differential enrichment of H3K79me2 upon NPM1 KD.Additional file 3: Table S2. List of primers used.