Rationale: Overexpression of NAD(P)H:quinone oxidoreductase 1 (NQO1) is associated with tumor cell proliferation and growth in several human cancer types. However, the molecular mechanisms underlying the activity of NQO1 in cell cycle progression are currently unclear. Here, we report a novel function of NQO1 in modulation of the cell cycle regulator, cyclin-dependent kinase subunit-1 (CKS1), at the G2/M phase through effects on the stability of c‑Fos.Methods: The roles of the NQO1/c-Fos/CKS1 signaling pathway in cell cycle progression were analyzed in cancer cells using synchronization of the cell cycle and flow cytometry. The mechanisms underlying NQO1/c-Fos/CKS1-mediated regulation of cell cycle progression in cancer cells were studied using siRNA approaches, overexpression systems, reporter assays, co-immunoprecipitation, pull-down assays, microarray analysis, and CDK1 kinase assays. In addition, publicly available data sets and immunohistochemistry were used to investigate the correlation between NQO1 expression levels and clinicopathological features in cancer patients.Results: Our results suggest that NQO1 directly interacts with the unstructured DNA-binding domain of c-Fos, which has been implicated in cancer proliferation, differentiation, and development as well as patient survival, and inhibits its proteasome-mediated degradation, thereby inducing CKS1 expression and regulation of cell cycle progression at the G2/M phase. Notably, a NQO1 deficiency in human cancer cell lines led to suppression of c-Fos-mediated CKS1 expression and cell cycle progression. Consistent with this, high NQO1 expression was correlated with increased CKS1 and poor prognosis in cancer patients.Conclusions: Collectively, our results support a novel regulatory role of NQO1 in the mechanism of cell cycle progression at the G2/M phase in cancer through effects on c‑Fos/CKS1 signaling. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a cytosolic reductase that plays an important role in cellular responses to oxidative stress in vitro and in vivo. Previous studies have disclosed protective effects of NQO1 on proteins that are independent of its enzymatic activity NQO1 protects cells against various cytotoxic quinones and oxidative stress and catalyzes the reduction and detoxification of quinone substrates, thereby preventing cytotoxic effects of carcinogens Previously, our group reported that NQO1 in cancer cells induces rapid degradation of Aurora-A during mitotic progression, specifically demonstrating that an NQO1 deficiency leads to aneuploidy during mitotic progression in irradiated cancer cells CKS1B gene on human chromosome Iq21, has a molecular weight of 9 kDa and is highly functionally conserved Cell cycle progression is a highly ordered process regulated by the oscillating expression of positive and negative factors Experiments from the current study support a novel role of NQO1 in the regulation of cell cycle progression at the G2/M phase in cancer cells. Taken together, our data suggest that NQO1 directly interacts with the DNA-binding domain (DBD) of c-Fos and inhibits its proteasome-mediated degradation, thereby increasing its stability. This NQO1-mediated increase in protein stability of c-Fos further promotes expression of CKS1 in cancer cells, resulting in increased cell proliferation and radioresistance. High-level expression of NQO1 is consistently correlated with elevated expression of CKS1 and poor survival in cancer patients.Gain-of-function and loss-of-function experiments were conducted to establish the effects of NQO1 on cancer cell proliferation. To this end, RKO human colorectal cancer cells were transfected with small inhibitory (interfering) RNA (shRNA) targeting NQO1 (RKO/pshNQO1) or control scrambled shRNA (RKO/pshCont). As shown in Figure To further ascertain the precise role of NQO1 in cancer cell cycle progression, we conducted a cell cycle analysis using cancer cells that were synchronized at the G1/S phase boundary via double-thymidine blocking and subsequently released to allow progression through the cell cycle. Cells were harvested at different time-points after release, as indicated, and analyzed by flow cytometry with propidium iodide Figure A,B. The Next, we focused on the potential regulatory effect of NQO1 on CDK1 in cancer cells. As shown in P-value < 0.01) in RKO/pshNQO1 cells compared with RKO/pshCont cells  is associated with cell cycle progression from G2 phase to M phase CKS1B to promote progression from G2 phase to M phase in cancer cells. An analysis of CKS1B mRNA and CKS1 protein levels in RKO/pshCont and RKO/pshNQO1 cells showed that NQO1 depletion induced a decrease in CKS1B mRNA and CKS1 protein expression in RKO cells , MIA PaCa-2 (pancreas), PC3 (prostate), and U87-MG (brain) cell lines . For these experiments, cells were transfected with either pCKS1B promoter-luc or control pRL-luc. Notably, luciferase activity was significantly inhibited in NQO1-knockdown RKO (RKO/pshNQO1) cells, whereas NQO1-overexpressing MDA-MB-231 cells exhibited elevated luciferase activity relative to NQO1-deficient MDA-MB-231 cells  induced significant downregulation of the AP-1 (activating protein-1) target genes, CTGF and CYR61, compared with the corresponding levels in RKO/pshCont cells, an effect that was further confirmed by quantitative polymerase chain reaction (qPCR) analysis  or transfection control (pRL-luc). TRE-mediated transcriptional activity was suppressed in NQO1-knockdown (RKO/pshNQO1) cells, whereas overexpression of NQO1 in MDA-MB-231 cells led to elevated TRE-luc activity compared with NQO1-deficient MDA-MB-231 cells  to promote CKS1B promoter using chromatin immunoprecipitation (ChIP) assays. To this end, DNA from RKO/pshCont cells and MDA-MB-231/pNQO1 cells incubated for 16 h was crosslinked, extracted, and incubated with anti-c-Fos antibody or control antibody (anti-IgG). c-Fos/DNA complexes were immunoprecipitated and crosslinking was reversed, followed by PCR amplification targeting TRE2 on CKS1B. Complexes immunoprecipitated with the anti-c-Fos antibody generated a PCR band, confirming association with TRE2 in the CKS1B promoter, whereas those obtained with the control antibody produced no PCR bands  and pCont, or sic-Fos and pCKS1B (CKS1B overexpression), and NQO1-deficient cancer cells were transfected with pCont and siCont, pc-Fos and siCont, or pc-Fos (c-Fos overexpression) and siCKS1B (CKS1B knockdown). After incubating for 48 h, cells were synchronized at the G1/S phase boundary using a double-thymidine block, then released for 9 h, harvested, and analyzed for levels of the indicated proteins , we established the stabilizing effects of NQO1 on c-Fos protein, demonstrating that NQO1 stabilized c-Fos by inhibiting its proteasomal degradation  database revealed significant correlation of elevated s Figure A-B. Exprs Figure . Immunohs Figure A,B. Furte Figure C,D. To es Figure E,F. CollNQO1 is a cytosolic reductase that exerts essential cytoprotective antioxidant effects by catalyzing the two-electron reduction of potentially toxic quinones, thereby preventing cytotoxicity of diverse carcinogens NQO1, together with genes encoding pentose phosphate pathway enzymes, ATP-binding cassette (ABC) transporter and some heme-metabolizing enzymes, is among the targets of nuclear factor (erythroid-derived 2)-like 2 (NRF2), which acts through the proteins encoded by its target genes to protect cells from intracellular and extracellular oxidative stress KEAP1 or NRF2 gene or other mechanisms that disrupt the binding of KEAP1 to NRF2 Suppression of NQO1 induces growth-inhibitory effects CKS1B encoding the CKS1 protein in cancer cells  and NQO1-deficient (RKO/pshNQO1) cells. Microarray analyses showed that an NQO1 deficiency led to a significant downregulation of the AP-1 target genes, CTGF and CYR61, in RKO/pshNQO1 compared with RKO/pshCont cells, an effect confirmed in MDA-MB-231 cells. AP-1, which is linked to cancer, is a dimeric transcription factor composed of proteins belonging to the Jun , Fos , and activating transcription factor (ATF) families CKS1B promoter, which contains two AP-1-like elements, using a promoter deletion assay and a reporter plasmid. This analysis showed that the sequence between positions -316 and -167 was required for NQO1-mediated AP-1 induction of CKS1B expression  that mediate n Figure . A furthn Figure G. Convern Figure H. Thus, n Figure I,J.‑/‑) and c-Fos (c-Fos-/-) genes -/- cells heterozygous or homozygous for the FosB gene  A previous report by Adler et al. demonstrated that NQO1 physically interacts with newly synthesized c-Fos in the cytoplasm and protects it from degradation until it engages in formation of an AP-1 complex and localizes to the nucleus An NQO1 deficiency in cancer cells leads to a delay in cell cycle progression at the G2/M phase through reduced c-Fos-mediated CKS1 expression Figures . BecauseNQO1 and CKS1B reported in TCGA colorectal and breast cancer datasets were higher in colorectal and breast cancer compared with normal tissues and that CKS1B expression was dependent on the level of NQO1 expression  and cultured in DMEM or RPMI medium. Cells were incubated at 37NQO1 and CKS1B gene expression were analyzed using TCGA available from Oncomine . High and low expression groups were defined as those with NQO1 and CKS1B levels above and below the mean value, respectively.Correlations between oC until use in the CDK1 kinase assay. CDK1 kinase activity was analyzed using a specific CDK1 kinase assay kit  according to the manufacturer's instructions.Cells were washed twice with PBS (pH 7.2), resuspended in PBS (pH 7.2) containing an inhibitor cocktail (Roche Applied Science), sodium orthovanadate, and sodium fluoride, sonicated four times using 10-second pulses on ice, and centrifuged at 14,000 g for 20 minutes. The supernatant fractions were collected, transferred into microcentrifuge tubes, and stored at -80in situ on the arrays. Biotinylated cRNA was prepared from 500 ng total RNA according to the standard Affymetrix protocol . Following fragmentation, 12 μg RNA was hybridized for 16 h at 45oC on a GeneChip Human Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450 and scanned using the Affymetrix GeneChip Scanner 3000 7G. Data were analyzed via Robust Multi-array Analysis (RMA) using Affymetrix default analysis settings and global scaling as the normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. Normalized and log-transformed intensity values were subsequently analyzed using GeneSpring GX 12.6 (Agilent Technologies). Fold change filters included the requirement for genes to be present in at a level of 200% of control for upregulated genes and lower than 50% of control for downregulated genes. Through hierarchical clustering, data were clustered into groups that behaved similarly across experiments using GeneSpring GX 12.6.1 (Agilent Technologies). The clustering algorithm was Euclidean distance average linkage.Total RNA was extracted from RKO/pshCont and RKO/pshNQO1 cells using TRIzol reagent (Invitrogen) in keeping with the manufacturer's instructions. The quality and quantity of RNA was assessed with an Agilent 2100 bioanalyzer system (Agilent Technologies). Gene expression was analyzed with GeneChip® Affymetrix Primeview array (Affymetrix) composed of over 530,000 probes representing ~20,000 well-characterized human genes. For each gene, eleven pairs of oligonucleotide probes were synthesized 6 , and anti-GFP . Secondary antibodies were obtained from the following sources: anti-rabbit AlexaTM Fluor 488 , anti-rabbit AlexaTM Fluor 594 , anti-mouse HRP , and anti-rabbit HRP . The antibodies used as negative controls for immunoprecipitation included normal mouse IgG  and normal rabbit IgG .Cycloheximide (CHX) and epoxomicin (EPX) were purchased from Calbiochem (Merck KGaA). Sodium orthovanadate, sodium fluoride, β-glycerophosphatate, thymidine, hyroxyurea, and nocodazole were acquired from Sigma-Aldrich, along with trifluoroacetic acid (TFA), ninhydrin, piperidine and acetic anhydride. N,N´-dimethylformamide (DMF), triisopropylsilane (TIS) and HPLC solvents were purchased from Acros Organics and diisopropylethyl amine (DIPEA) and 3,6-dioxa-1,8-octanedithiol from TCI. Rink Amide MBHA resin was acquired from Advanced Chem Tech. 1-Hydroxybenzotriazole (HOBt) and o-(benzotriazol-1-yl)-N,N,N´,N´-tetramethyluronium hexafluorophosphate (HBTU) and N-α-Fmoc-protected amino acids (Novabiochem) were used as received. Antibodies were obtained from the following sources: anti-cyclin B1 , anti-NQO1 , anti-β-actin , anti-CDK1 , anti-CKS1 , anti-c-Fos , anti-c-JUN , anti-HisCKS1B promoter (ppro#-luc), total DNA was extracted from RKO cells using the AccuPrep® Genomic DNA extraction kit (Bioneer). The promoter of CKS1B was amplified via PCR with the appropriate primer pairs as follows: pro1, 5'-GGG GTA CCT CCC ACA AAG ATA AAG CTC-3' (forward) and 5'-GGA ATT CTC ATT TCT TTG GTT TCT TGG G-3' (reverse); pro2, 5'-GGG GTA CCT CAA CAA ATT CGA ATC GTT C-3' (forward) and 5'-GGA ATT CTC ATT TCT TTG GTT TCT TGG G-3' (reverse); pro3, 5'-GGG GTA CCC CGA GGA TCG TTG CTA G-3' (forward) and 5'-GGA ATT CTC ATT TCT TTG GTT TCT TGG G-3' (reverse); pro4, 5'-GGG GTA CCG GAC TTC CAG AAA AAC TGG-3' (forward) and 5'-GGA ATT CTC ATT TCT TTG GTT TCT TGG G-3' (reverse); pro5, 5'-GGG GTA CCG TCT CAG ACT TAT AAA TGA AG-3' (forward) and 5'-GGA ATT CTC ATT TCT TTG GTT TCT TGG G-3' (reverse); pro6, 5'-GGG GTA CCT ATT ACA CTC ACT TCC GGC-3' (forward) and 5'-GGA ATT CTC ATT TCT TTG GTT TCT TGG G-3' (reverse); pro7, 5'-GGG GTA CCG CTC GTT CTT GAG AAG CG-3' (forward) and 5'-GGA ATT CTC ATT TCT TTG GTT TCT TGG G-3' (reverse); △TRE1, 5'-AAG TCA AGA CTC ACC AGG-3' (forward) and 5'-CTT CCG GTC TTC ATT TAT AAG-3' (reverse); △TRE2, 5'-CGA GCT CCG CTC GTT CTT GAG AAG C-3' (forward) and 5'-CGA GCT CAA AGT AGG CGT CTT ATT GGC-3' (reverse); TRE1, 2△ 5'-CGA GCT CCG CTC GTT CTT GAG AAG C-3' (forward) and 5'-CGA GCT CCT TTC ACC CCG GAA CCC-3' (reverse) (Bioneer). PCR products were digested with the appropriate restriction enzymes and directly ligated into pGL3-basic (Invitrogen). To construct plasmids expressing NQO1 and the EGFP-c-FOS fusion protein, total RNA was obtained from RKO cells using TRIzol reagent (Invitrogen) and cDNA generated using SuperScriptTM III Reverse Transcriptase (Invitrogen). The open reading frames (ORF) of NQO1 and c-FOS were amplified via PCR with the appropriate primers as follows: NQO1, 5'-GGG GTA CCA TGG TCG GCA GAA GAG CAC-3' (forward) and 5'-CCG CTC GAG TTT TCT AGC TTT GAT CTG G-3' (reverse); c-FOS, 5'-CGG GAT CC ATG ATG TTC TCG GGC TTC AAC-3' (forward) and 5'-GGA ATT C CAG GGC CAG CAG CGT GG-3' (reverse); aa1-140, 5'-GGA ATT CTA TGA TGT TCT CGG GCT TCA AC-3' (forward) and 5'-GGG GTA CCC CTT TTC TCT TCT TCT TCT GG-3' (reverse); aa137-164, 5'-GGA ATT CTG AAG AGA AAA GGA GAA TCC G-3' (forward) and 5'-GGG GTA CCT GTA TCA GTC AGC TCC CTC-3' (reverse); aa161-198, 5'-GGA ATT CTC TGA CTG ATA CAC TCC AAG-3' (forward) and 5'-GGG GTA CCT GCC AGG ATG AAC TCT AG-3' (reverse); aa196-380, 5'-GGA ATT CTA TCC TGG CAG CTC ACC G-3' (forward) and 5'-GGG GTA CCC AGG GCC AGC AGC GTG-3' (reverse) (Bioneer). Amplified products were digested with restriction enzymes and directly ligated into pCDNA3.1-myc-His6 (Invitrogen) or pEGFP-c1 (Clontech) vectors for cloning. The cloned plasmids were analyzed via restriction digestion and DNA sequencing (Bionics). To construct stable cell lines, cells were seeded at a density of 5 × 104 cells per well in 24-well plates and transfected with 50 μl mixture containing 1 μg pCDNA3.1-NQO1-myc-His6 (Invitrogen) or pshNQO1 (Qiagen) along with TurboFect in vitro transfection reagent (Fermentas). pCDNA3.1-myc-His6 (pCont) and pshCont (Qiagen) were utilized as controls. Transfected cells were selected with 1 mg ml-1 G418 or 1 μg ml-1 puromycin (Duchefa Biochemie) for 1 week and maintained in DMEM or RPMI-1640 containing 0.5 mg ml-1 G418 or 0.3 μg ml-1 puromycin during the experiments.To construct a luciferase reporter plasmid encoding 4) were seeded into 25 cm2 flasks, incubated overnight, and co-transfected with 50 μl mixture containing 1 μg 3 × AP1pGL3 , ppro#-luc or pCKS1B promoter-luc and 0.01 μg pRL-luc  using the TurboFect in vitro transfection reagent (Fermentas). After 4 h, cells were washed with PBS and incubated with the appropriate medium for 48 h. Luciferase activity was determined using a luciferase assay system (Promega) and normalized with respect to Renilla luciferase activity according to the manufacturer's instructions. Three independent transfections were performed in each case.Cells .Total RNA was extracted from RKO and MDA-MB-231 cells using TRIzol reagent (Invitrogen) and treated with DNase I (New England Biolabs). Next, total RNA (1 μg) was used for cDNA synthesis with AccuPower RT PreMix , and the resulting cDNA amplified via PCR with the following primer pairs: NQO1, 5'-CCC TGC GAA CTT TCA GTA TCC-3' (forward) and 5'-CTT TCA GAA TGG CAG GGA CTC-3' (reverse); overexpressed NQO1, 5'-TTG ACC TAA ACT TCC AGG C-3' (forward) and 5'-TAG AAG GCA CAG TCG AGG CTG-3' (reverse); CTGF, 5'- TCC CGA GAA GGG TCA AGC T-3' (forward) and 5'- TCC TTG GGC TCG TCA CAC A-3' (reverse); CYR61, 5'-TCC TCT GTG TCC CCA AGA AC-3' (forward) and 5'-TCG AAT CCC AGC TCC TTT ACC-3' (reverse) (Bioneer). qPCR was performed using iQ SYBR Green Supermix (2x) (Bio-Rad) and analyzed with the CFX Connect2+1684.94, found 1685.01, m/z calculated for [M+3H]3+ 1123.63, found 1123.68, m/z calculated for [M+4H]4+ 842.97, found 843.01.The NQO1-binding c-Fos peptide (Ac-EEKRRIRRER NKMAAAKCRNRRRELT-NH2) was synthesized using Fmoc-chemistry in solid phase peptide synthesis 6- and EGFP-tagged proteins were overexpressed in MDA-MB-231 cells. Cells were lysed using ice-cold RIPA/PBS (33% v/v) containing an inhibitor cocktail (Roche Applied Science), sodium orthovanadate, and sodium fluoride. Total cell protein (1 mg) was incubated with 25 μl washed Protein G-magnetic beads (New England Biolabs) at 4°C for 1 h. Cleared lysates were incubated overnight at 4°C with 5 μg mouse monoclonal His6, mouse monoclonal GFP (Roche) or normal mouse IgG antibody, followed by 25 μl washed Protein G-magnetic beads at 4°C for 1 h. The immunoprecipitation matrix-antibody complex was washed three times with ice-cold RIPA/PBS (33% v/v), and bound proteins resolved via SDS-PAGE and subjected to immunoblot analysis. Signals were detected using enhanced chemiluminescence (Pierce). For endogenous co-immunoprecipitation, cells were lysed using ice-cold RIPA/PBS (33% v/v) containing an inhibitor cocktail (Roche Applied Science), sodium orthovanadate, and sodium fluoride. Rabbit monoclonal c-FOS, rabbit monoclonal c-JUN, normal mouse IgG and normal rabbit IgG antibodies (5 μg) were used for these experiments. Co-immunoprecipitation and immunoblot analyses were performed as described above. Uncropped images of the blots are shown in For co-immunoprecipitation, HisCKS1B promoter with the following primer pairs: PCR was performed with the following primer pairs: 5'-TTA GCC AAT GGC AGC GCG AGA TC-3' (forward) and 5'-TCC TTA TTG GAG GGA GTT CTC-3' (reverse). The PCR products were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide (EtBr) (Sigma-Aldrich).ChIP assays were performed using the ChIP Assay Kit (Sigma-Aldrich) according to the manufacturer's protocol. Briefly, cancer cells incubated in a T25-flask were crosslinked by treatment with formaldehyde  for 10 min at room temperature. After washing with PBS, cells were pelleted and resuspended in SDS lysis buffer , 1 mM DTT, and 1 mM PMSF). The lysates were then subjected to sonication to reduce the DNA length to between 500 and 1000 bp, diluted with dilution buffer , 167 mM NaCl), and pre-cleared by incubating with a Salmon Sperm DNA/protein A agarose-50% slurry for 60 min at 41C. The supernatant was incubated with anti-c-Fos  or anti-Rabbit normal IgG  at 4°C overnight. Immunocomplexes were collected with the Salmon Sperm DNA/protein A agarose-50% slurry and eluted after extensive washings, and crosslinking was reversed by heating at 65°C, followed by treatment with 40 mg/ml proteinase K at 45°C for 60 min. DNA was recovered by phenol-chloroform/ ethanol precipitation, and was used as a template for PCR to amplify the target sites in the -1 G418 for one week, and maintained in DMEM or RPMI-1640 containing 0.5 mg ml-1 G418. NQO1-expressing RKO (RKO/pshCont) and MDA-MB-231 (MDA-MB-231/pNQO1) cells were transfected with siCont or sic-Fos and NQO1-deficient RKO (RKO/pshNQO1) and MDA-MB-231 (MDA-MB-231/pCont) cells transfected with pCont or pc-FOS. After a 24 h incubation period, NQO1-expressing cells were transfected with pCont or pCKS1B and NQO1-deficient cells were transfected with siCont or siCKS1B. After further 24 h incubation, cells were transfected with pGCGFP  or pimEJ5GFP . Two days after transfection, cells were subjected to flow cytometry using the BD FACSMelodyTM instrument combined with a cell sorter (BD Biosciences). Three independent transfections were performed for each case.HR and NHEJ reporter assays were performed as previously described 6- and EGFP-tagged proteins were expressed in MDA-MB-231 cells and RKO cells, respectively. His6-proteins were affinity-purified and subsequently conjugated to Ni-NTA resin (Qiagen). His6-conjugated resin was incubated for 2 h at 4oC with 1 mg total protein of RKO cells. The resin was extensively washed, eluted, and subjected to immunoblot analysis.HisNQO1, CKS1B and c-Fos were subjected to RNA interference using a 19 bp (including a 2-deoxynucleotide overhang) small interfering RNA (siRNA). siRNAs against NQO1 (CCGUACACAGAUACCUUGAdTdT), CKS1B (GUGACUUGCGGAUUUAUGUdTdT) and c-Fos (GUAUCUAGUGCAGCUGAUUdTdT) were purchased from Bioneer Corporation , with StealthTM RNAi (Invitrogen) used as a negative control. Cells were seeded in 25 cm2 flasks, grown to ~80% confluence, and transfected with siRNA duplexes using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol. After 48 h, cells were processed for analysis as indicated.oC and 5% CO2 to promote colony formation. The culture medium was decanted, and colonies were fixed with 95% methanol and stained with 0.5% crystal violet. The numbers of colonies (>50 cells) from triplicate dishes or plates were counted and the mean number of colonies formed by irradiated cells compared with that formed by untreated cells.Varying numbers of cells were plated on 6-well plates and irradiated (4 Gy) or left untreated. Cells were incubated for 14 days at 37Cells were exposed to X-rays using an X-RAD Ir160 X-ray irradiator (Accela)Coverslip-mounted cells were fixed with 3.7% paraformaldehyde (PFA) and incubated in blocking solution (3% BSA) for 1 h at room temperature, after which they were incubated overnight at 4ºC with anti-phospho histone H2AX (ser139) , anti-c-Fos  or anti-NQO1  antibodies. Cells were washed three times with blocking solution and incubated with Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies for 1 h. After washing twice with PBS, cells were stained with DAPI (Invitrogen) for 10 min to stain nuclei. Coverslips were washed three times with PBS and mounted onto slides using mounting reagent (Invitrogen), followed by analysis using laser-scanning confocal microscopy .Cells were synchronized at the G1/S boundary using the double-thymidine block method. Cells were initially treated with 2 mM thymidine for 18 h, incubated in fresh medium without thymidine for 8 h, and retreated with thymidine for a further 16 h. Cells were finally released from arrest by replacing thymidine solution with fresh medium. For the G2/M boundary synchronization, cells were treated with 100 nM nocodazole for 22 h and released from the block by incubation with fresh medium. For analysis of CDK1 kinase activity and expression of cyclins and CDK, immunoblot analysis and CDK1 kinase assays were performed as described above. For cell cycle analysis, cells were fixed in cold 70% (vol/vol) ethanol, washed with cold phosphate-buffered saline (PBS) and stained with 40 μg/ml propidium iodide in the presence of 50 μg/ml ribonuclease A for 30 min at room temperature. Cellular DNA (10 000 cells per sample) was analyzed via flow cytometry (BD Biosciences).2O2 in absolute methanol for 15 min at room temperature. Sections were incubated overnight at 4°C with anti-NQO1  or anti-CKS1  antibody and washed with PBS containing 0.05% Trion X-100. Incubation with a secondary antibody and the peroxidase-antiperoxidase (PAP) complex was carried out for 30 min at room temperature. Immunoreactive sites were visualized using 3,3-diaminobenzidine tetrachloride . Slices were subsequently counterstained with hematoxylin.Human colon cancer and human breast cancer tissues were obtained from US Biomax . Paraffin-embedded sections were deparaffinized and rehydrated. Immunohistochemical analysis of NQO1 and CKS1 was performed with a Vectastain Elite ABC kit  in keeping with the manufacturer's protocol. For antigen retrieval, sections were placed in citrate buffer (pH 6.0) and heated in a microwave oven for 10 min. For immunoperoxidase labeling, endogenous peroxidase was blocked with 0.3% HAll grouped data are presented as mean ± SD. Differences between groups were analyzed with ANOVA or Student's t-test using GraphPad Prism software. For survival analysis, Kaplan-Meier curves were generated using Prism software and log-rank analysis performed. All experiments were conducted at least in duplicate with three technical replicates.Supplementary figures and table.Click here for additional data file.


Arabidopsis.Heritable base-editing using a viral delivery system enables high-throughput functional analysis of genes in  Dear Editor,Arabidopsis . Transgenic Arabidopsis plants expressing base-editing reagents are often somatic mosaics in the first generation, and multiple generations are required to fix edited alleles (Arabidopsis lines expressing a cytidine deaminase base-editor (Arabidopsis UBIQUITIN 10 (UBI10) promoter, is a Cas9 nickase fused to the human APOBEC3A (hA3A)/Y130F cytidine deaminase (PHYTOENE DESATURASE 3 (PDS3) and CLOROPLASTOS ALTERADOS 1 (CLA1) were recovered at frequencies of 7.66% and 2.73%, respectively. Up to 8.90% of progeny from one plant were homozygous for mutations (C to T) in PDS3 and exhibited an albino phenotype caused by introduction of a premature stop codon. Plants with gain-of-function mutations that confer herbicide tolerance (both homozygous and heterozygous) were recovered in CELLULOSE SYNTHASE 3 (CESA3) at a frequency of 2.28%. These results suggest that virus-mediated base-editing can be performed at scale in Arabidopsis, allowing high-throughput functional analysis of genes through precise mutagenesis.Precise base-editing technologies have great potential for studying plant gene function and accelerating crop improvement . To date alleles . Recentl alleles . Here, we-editor . The baseaminase . ProgenyArabidopsis lines (L4 and L8) that highly express the APOBEC3A-derived C-to-T base-editor, using reverse transcription quantitative PCR (RT-qPCR)  vectors, the esgRNAs were fused at their 3′-ends to a tRNA isoleucine (tRNAIleu). We and others have previously shown that tRNAs and other RNAs that promote cell-to-cell movement can increase virus-mediated mutagenesis when fused to sgRNAs  . Enhanceith Cas9 . Mutagenectively . Disruptynthesis . CLA1 ishenotype . Prior to sgRNAs . The TRVg method . The infe leaves . Base-edPDS3 esgRNA from both L4 and L8 grown at 18°C and 16-h light resulted in plants with pronounced bleached spots in rosette leaves  typically lasts for about three weeks . Growinge leaves . As the e leaves . When the leaves . The knoectively .PDS3 esgRNA and CLA1 esgRNA vectors at frequencies of 3.55% and 0.79%, respectively  albino progeny -7-(2-methoxyphenyl)-1,5,6,7-tetrahydro-triazolo pyrimidine) (UBIQUITIN 4-2 promoter from parsley (Petroselinum crispum) and the CESA3 sgRNA to create C17 tolerance; however, no homozygous mutants were recovered in approximately 2,000 T1 seedlings tested. Homozygous base-editing events were recovered in subsequent generations  in the M0 plants. Seeds from infected parental plants were collected and directly sown on 0.5 MS medium supplemented with 1 μM C17 according to the method of CESA3_P3  . In a prerations . Here, wCESA3_P3 . Primersing Cas9 . MutagenArabidopsis floral dip transformation method has been previously used to deliver base-editing reagents, germline edits were difficult to attain. The recovery of homozygous mutants required screening progeny through several generations  (unpublished)—and probably can be expanded to other plant species. Indeed, recent work with Barley Stripe Mosaic Virus vectors demonstrated heritable editing through infection in wheat (Triticum aestivum)  . The simThe following materials are available in the online version of this article. TRV vectors, base-editor reagents, and the expression of the base-editor in transgenic Arabidopsis plants. Virus-mediated base-editing in Arabidopsis under a 24°C/22°C and 12-h/12-h day/night cycle. Pronounced pds3 knockout phenotype at an early developmental stage in M0 Arabidopsis plants. Representative base-editing profiles in cauline leaves from plants grown at 18°C with a 16-h/8-h day/night cycle. Mosaic phenotypes of 8-day-old PDS3 base-edited M1 seedlings. The frequency of heritable base-editing at PDS3 and CLA1. The frequency of heritable base-editing at CESA3. Primers used in this study.kiac206_Supplementary_DataClick here for additional data file.


C. elegans. Using a photoconvertible fluorescent protein-based imaging assay, we found that SEIP-1 positive LDs were selectively depleted after fertilization, coincident of the formation of a lipid-rich permeability barrier of the eggshell. Loss of SEIP-1 function caused impenetrant embryonic arrest, which could be worsened by FAT-3/fatty acyl-CoA desaturase deficiency or suppressed by PLIN-1/Perilipin deficiency. The embryonic development of seip-1; plin-1 mutant in turn depended on the recruitment of RAB-18/Rab18 to LDs, which was not observed in wild type embryos. We propose that SEIP-1 dependent and independent mechanisms act in parallel to ensure the packaging and export of lipid-rich permeability barrier constituents, which involve LDs. The identity of these LDs, as defined by their associated proteins, exhibits unexpected plasticity that ultimately ensures the survival of embryos ex utero.In addition to coordinating the storage and mobilization of neutral fat, lipid droplets (LDs) are conserved organelles that can accommodate additional cargos in order to support animal development. However, it is unclear if each type of cargo is matched with a specific subset of LDs. Here, we report that SEIP-1/seipin defines a subset of oocyte LDs that are required for proper eggshell formation in  The core of LDs contains neutral lipids, such as triacylglycerol (TAG) or cholesterol ester. It is known that the composition of the neutral lipid core varies in a cell type- and nutrient-dependent manner, which in part reflects the demand and supply of specific lipid species , which are evolutionarily conserved organelles that coordinate fat storage and utilization, are readily detected in vertebrate and invertebrate oocytes and embryos . The str species .C. elegans intestinal cells associate with a tubular ER-subdomain, defined by the preferential enrichment of the seipin ortholog, SEIP-1  deletion allele [seip-1(-) thereafter] was initially annotated as lethal by the National Bioresource Project (seip-1(-) worms were fertile   worms, we cultured them in the presence of the vital dye BODIPY on standard nematode growth media (NGM) plates. Although the total number of eggs laid by wild type and seip-1(-) worms were comparable (seip-1(-) eggs were stained (seip-1(-) embryos develop and hatch as L1 larvae, we therefore conclude that SEIP-1 acts in parallel of additional proteins to ensure embryonic viability. Interestingly, seip-1(-) eggs laid by relatively young adults were more prone to BODIPY staining , the chitin layer (CL), the chondroitin proteoglycan layer (CPG), and the permeability barrier were sequentially formed toward the embryonic plasma membrane (seip-1(-) worms was similar to a class of mutants that fail to form the permeability barrier properly. Accordingly, a large number of perm-1 and dgtr-1 embryos could be stained when they were laid on BODIPY-containing NGM plates (seip-1(-) embryos  embryos, whereas the exocytosis of CPG-2 was unperturbed  or SEIP-1 (A185P)::GFP (hjSi541), driven by the ubiquitous dpy-30 promoter. Wild type SEIP-1::GFP supported the formation of the embryonic permeability barrier (seip-1(-) mutants (seip-1(-) mutant worms (seip-1(A185P) allele (hj158) using CRISPR. Based on our homology directed repair (HDR) methodology, an “orphan” loxP sequence was inserted between the stop codon and 3’-UTR of seip-1(A185P)  with a loxP site inserted at the same position as in hj158 (seip-1(hj156) animals was comparable to that of wild type. However, the fertility of seip-1(hj158)  was reduced to a level similar to seip-1(-) animals, with a corresponding increase in BODIPY-positive embryos (The Alanine 212 to Proline (A212P) mutation in human seipin causes congenital generalized lipodystrophy type 2 disease (CGL2) . Based o barrier  and rest1(A185P) . We therin hj158 . The fer embryos . Our obs embryos . We concC. elegans, the proper embryonic development is dependent on the supply of intestine-derived yolk proteins (sun-1 promoter (hjSi502) fully rescued the defect of seip-1(-) embryos (seip-1(-) worms to the wild-type level (vha-6 promoter (hjSi3) did not rescue the embryonic defect (seip-1(-) mutants (seip-1(-) embryos  worms. Because DGAT-2 is primarily found in intestinal cells, we needed an alternative marker to visualize germline LDs. To this end, we used the sole C. elegans perilipin ortholog, MDT-28/PLIN-1 , which is ubiquitously expressed (hj178) for which the two long isoforms of PLIN-1 (PLIN-1a/c) were tagged with GFP at their C-termini (seip-1(-) worms were smaller than those in wild type worms (seip-1(-) mutants (seip-1(-) germline was larger than that in wild type (seip-1(A185P) loss of function mutants .We previously demonstrated that SEIP-1 regulates intestinal LD expansion , using t, DGAT-2 , as the xpressed . We engi-termini . Consistpe worms , inset ipe worms , we foun mutants . As a reild type . We made mutants . Such ab mutants . Our resC. elegans intestine. To investigate the localization of SEIP-1 in the germline, we first examined SEIP-1(WT)::GFP in adult worms that lacked the endogenous SEIP-1 protein. In both oocytes and embryos, SEIP-1(WT)::GFP was targeted to “ring” or “cage”-like structures (seip-1(-) worms that expressed human seipin::GFP ubiquitously (hjSi434), was functional since it could rescue both the fertility and the permeability barrier defect of seip-1(-) worms  and COPII vesicles (SEIP-1 is enriched at a subdomain of the endoplasmic reticulum (ER), termed the peri-LD cage , in the ructures . Similaruitously . In compuitously . Such louitously . Next, w-) worms . SEIP-1:-) worms . Therefo-) worms  and yolk-) worms  are know-) worms . They cavesicles . Accordivesicles . For thevesicles . Overallvesicles . Our resWe next sought to understand how SEIP-1 (+) LDs might regulate the formation of the permeability barrier. Based on live imaging, we consistently observed LDs and SEIP-1-labeled peri-LD cages near the cell cortex of +1 embryos, but not in −1 oocytes or +2 embryos . To quanseip-1 locus, we expressed the fusion of the monomeric photoconvertible fluorescent protein mKikGR (We hypothesized that the redistribution of SEIP-1 (+) LDs near the plasma membrane supports the construction of the permeability barrier in newly fertilized embryos. To determine if such redistribution is linked to the catabolism or anabolism of SEIP-1 (+) LDs, we developed a photoconversion strategy to label preexisting SEIP-1 in the oocytes. From the endogenous n mKikGR  with SEIn mKikGR . These wn mKikGR , fertilin mKikGR . Therefon mKikGR , endogenn mKikGR , inset in mKikGR . In thisA genetic pathway, consisting of FASN-1, POD-2, PERM-1, and DGTR-1, has previously been shown to control permeability barrier formation . How doeseip-1(-) worms (seip-1(-) worms (seip-1(-) worms. Indeed, FAT-3 deficiency reduced the fertility of otherwise wild type worms or seip-1(-) worms (seip-1(-); fat-3(-) worms displayed a highly penetrant embryonic arrest phenotype, similar to dgtr-1 or perm-1 deficient worms. We conclude that FAT-3 products and their derivatives are important for both SEIP-1-dependent and SEIP-1-independent pathways that are responsible for permeability barrier formation. Both pathways are blocked in seip-1(-); fat-3(-) worms.We next sought to understand the molecular basis of incomplete penetrance and high variability of the embryonic defect of -) worms . FAT-3, -) worms  and oocy-) worms . Thus, F-) worms , suggest-) worms . Accordiseip-1(-) background, ubiquitous depletion of either fat-3 or fat-4 increased the number of embryos with permeability barrier defects (seip-1(-) mutants. Similar effect was not observed when FAT-1 or FAT-5 was depleted  synthesis and desaturation . The per defects . Accordidepleted .seip-1(-) worms, we repeated our experiments with strains that restricted RNAi to the intestine or germline (seip-1(-) mutant background, intestine-specific depletion, but not germline-specific depletion, of FAT-2, FAT-3, or FAT-4 enhanced the embryonic arrest phenotype  worms, different PUFAs and their derivatives appear to contribute to germline and embryonic development in multiple ways, including but not limited to the establishment of ER subdomains and intestine-to-germline lipid transport. The distribution of these PUFA derivatives in specific membranes and vesicles will require further chemical analysis.To determine the tissues from which fatty acyl-CoA desaturases modulated the phenotypes of germline . In seiphenotype . Therefown worms , suggestwn worms . Howeverseip-1(-) worms. In an attempt to tag all isoforms of PLIN-1 with the mRuby red fluorescent protein, we inserted the mRuby coding sequence to the 5’ end of the plin-1 gene by CRISPR  worms, we were surprised that the fertility of plin-1(hj249); seip-1(-) worms was similar to that of wild type worms (plin-1(hj178) when PLIN-1A and PLIN-1C isoforms were fused at their C-terminus to GFP  deletion allele (plin-1(-) hereafter). The plin-1(-) worms showed normal permeability barrier formation and fertility (plin-1(hj249); seip-1(-) worms, the fertility of plin-1(-); seip-1(-) worms was comparable to that of wild type worms, and almost all embryos had an intact permeability barrier as indicated by the lack of BODIPY staining (plin-1(-) allele rescued the embryonic defects of seip-1(A185P) mutants (plin-1(-) was not restricted to specific seip-1 loss of function alleles.We inadvertently discovered that PLIN-1 inhibited an alternative mechanism that supported permeability barrier formation in y CRISPR . When wepe worms . Accordipe worms . These ps to GFP . Such din allele   (plin-1 did not show similar suppression (plin-1 in the germline of plin-1(-); seip-1(-) worms, with a sun-1 promoter driven single-copy transgene. These worms showed embryonic defects similar to seip-1(-) worms (fat-2 or fat-3 and intestine-specific knockdown of fat-3 by RNAi reversed the suppression of seip-1(-) associated embryonic defects by plin-1(-) (plin-1(-); seip-1(-) worms appeared to rely on FAT-2 or FAT-3, acting in distinct tissues, to support permeability barrier formation. Our results support the notion that local and diffusible PUFAs and their derivatives are required for embryonic development of C. elegans.Next, we used RNAi to knockdown eip-1(-) . Howeverpression . As a co-) worms . Thus, olin-1(-) . Thereforab-18 knockdown yielded BODIPY-positive, permeability barrier defective plin-1(-); seip-1(-) embryos ; seip-1(-) embryos (dgtr-1 function caused highly penetrant embryonic arrest of otherwise wild type worms (rab-18 (ok2020) [referred to as rab-18(-) hereafter] loss of function allele did not arrest as embryos and produced comparable number of live progenies as wild type worms (rab-18 significantly increased the percentage of arrested embryos from plin-1(-); seip-1(-) worms and reduced the number of live progenies accordingly ; plin-1(-); rab-18 (S25N) mutants shared the same permeability barrier defect as plin-1(-); seip-1(-); rab-18(-) mutants (plin-1(-); seip-1(-) worms to support embryonic development.Next, we sought to identify proteins that acted redundantly with SEIP-1 to ensure embryonic development. To this end, we inactivated genes encoding known or putative LD-associated proteins by RNAi. We found that ubiquitous or germline-specific  embryos . Intestio effect . Similarormation , was kno embryos . Our respe worms . In contpe worms . Howeverordingly . Using Cmutation . The S25 mutants . Our resplin-1(-) worms (seip-1(-) worms, GFP::RAB-18 appeared in numerous diffraction-limited puncta throughout the cytoplasm of newly fertilized  embryos (plin-1(-); seip-1(-) embryos, GFP::RAB-18 was found on the LD surface as confirmed by the LDP-1::mRuby marker (seip-1(-) embryos similarly permitted the association of RAB-18 with LDs that contained the cargos for permeability barrier construction. Taken together, our results support the notion that RAB-18 positive LDs assume an identity that is similar to SEIP-1 (+) LDs in otherwise wild type embryos. The association of RAB-18 with LDs is modulated by other resident LD proteins, such as PLIN-1.To elucidate the mechanism by which RAB-18 supported permeability barrier formation in the absence of SEIP-1 and PLIN-1, we investigated the localization of GFP::RAB-18 fusion protein, expressed from its endogenous locus . It did -) worms . In seip embryos . Finallyy marker  ; seip-1(-) embryos. Using multiple independent deletion alleles (plin-1(-); seip-1(-) embryos (plin-1(-); seip-1(-); atgl-1(-) embryos was lower than that of plin-1(-); seip-1(-); rab-18(-) embryos. We also noted that ATGL-1 was already present on lipid droplets in plin-1(-) single mutants (plin-1(-); seip-1(-) mutants (plin-1(-); seip-1(-) mutants.Because perilipin orthologs are best known for their role in regulating lipolysis , we aske alleles , we foun embryos . However mutants , whereas mutants . Taken tC. elegans eggshell. Our results suggest that SEIP-1 (+) LDs contribute to the packaging and release of lipid-rich ascarosides that are eventually exported to the embryonic extracellular space. This hypothesis is compatible with the model that embryonic cortical granules are responsible for the export of additional material that constitutes the protein-rich layers of the eggshell (seip-1(-) embryonic arrest phenotype suggests that parallel pathways exist to permit ‘alternative’ LDs to accommodate and release ascarosides : LG III, unc-119 (ed3), rab-18 (ok2020), itIs38 [(pAA1)pie-1p::GFP::PH (PLC1delta1) + unc-119(+)]; LG IV, fat-3 (ok1126); LG V, sid-1 (qt78), ltIs44 [(pAA173)pie-1p::mCherry::PH (PLC1delta1) + unc-119 (+)]; LG X, vit-2 (crg9070). seip-1 (tm4221) V and plin-1 (tm1704) I alleles were obtained from Dr. Shohei Mitani . rde-1 (mkc36) V and mkcSi13 [sun-1p::rde-1::sun-1 3′UTR] II are gifts from Dr. Di Chen . The following single-copy transgenes were used: hjSi3 [vha-6p::seip-1 cDNA::GFP_TEV_3x- FLAG::let-858 3′UTR] II, hjSi189[dpy-30p::seip-1 cDNA:: GFP_TEV_3xFLAG::tbb-2 3′UTR] II, hjSi206 [vha-6p::human seipin isoform 2 cDNA (codon optimized)::GFP_TEV_3xFLAG::let-858 3′UTR] II, hjSi223[dpy-30p::human seipin isoform 2 cDNA (codon-optimized)::GFP_TEV_3xFLAG::tbb-2 3′UTR] II, hjSi434[dpy-30p::seip-1 cDNA::tagRFP::tbb-2 3′UTR] II, hjSi494[vha-6p::sid-1 cDNA::dhs-28 3′UTR] I, hjSi502[sun-1p::seip-1 cDNA::GFP_TEV_3xFLAG::sun-1 3′UTR] II, hjSi541[dyp-30p::seip-1(A185P) cDNA::GFP_TEV_3xFLAG::tbb-2 3′UTR] II, hjSi552[sun-1p::plin-1 gDNA::GFP_TEV_3xFLAG::sun-1 3′UTR] II. The following CRISPR/Cas9-generated alleles were used: hj140[seip-1::GFP_TEV_3xFLAG] V, hj158[seip-1(A185P)::stop codon_loxP_seip-1 3'-UTR] V, hj178[plin-1a/c::GFP_TEV_3xFLAG] I, hj249[mRuby_TEV_3xFLAG::plin-1] I, hj256[sec-16A.1::GFP_TEV_3xFLAG] III, hj289[ldp-1::mRuby_TEV_3xHA] V, hj323[seip-1::mKikGR_SEC_3xFLAG] V, hj340[cpg-2 signal peptide::mCherry_TEV_3xFLAG::cpg-2 w/o signal peptide] III, hj345[GFP_TEV_3xFLAG::atgl-1] III, hj347[GFP_TEV_3xFLAG::rab-18b] III, atgl-1  III, lid-1  I, hosl-1  X, hj371[GFP_TEV_3xFLAG::rab-18b(S22N)] III. The sgRNA sequences for CRISPR are listed in Bristol N2 was used as the wild-type hjSi494) or germline-specific (mkcSi13) transgene is used to rescue sid-1(qt78) or rde-1(mkc36) mutant animals, respectively. The seip-1(tm4221) and/or plin-1(tm1704) allele were introduced into hjSi494; sid-1 (qt78) and mkcSi13; rde-1(mkc36) by genetic crosses. Further details of the RNAi clones can be found in RNA interference (RNAi) was performed by on-plate feeding according to published methods . The tarFor all fluorescence imaging, one-day-old adult animals were examined by a spinning disk confocal microscope  with a ×5  0.16) or ×63  1.4 oil) Alpha-Plan-Apochromat objective. Image stacks were acquired with a Neo sCMOS camera (Andor) and a piezo Z stage controlled by the iQ3 software (Andor). For GFP, a 488 nm laser was used for excitation and emitted signals were collected by a 500–550-nm filter. For mCherry, mRuby, tagRFP, and photo-converted mKikGR, a 561 nm laser was used for excitation and emitted signals were collected by a 580.5–653.5-nm filter. Optical sections of images at 0.5 μm intervals were exported to Imaris 8 (Bitplane) for processing and 3D reconstruction.C. elegans, fresh 8% agarose pads were prepared on top of microscope glass slides. 2–3 μl of 1.25% (w/v) polystyrene microspheres  and 0.2 mM levamisole (Sigma) in 1 × PBS were dropped at the center of the agarose pads for immobilization of adult animals. For imaging the permeability barrier, on 22 mm × 22 mm cover slips, embryos were dissected from the uterus of one-day-old adults  in 0.8 × egg buffer with 0.2 mM levamisole in spherical micro-chambers bordered by vaseline. The microscope glass slides were subsequently applied with care to avoid the compression of the embryos.For imaging the germline and embryos of live seip-1(hj323) adults  were exposed to 400–440 nm fluorescence filtered by SZX2-FBV (Olympus) for 30 min on a stereomicroscope  equipped with a LED light source (EXFO). Worms were allowed to rest for 1 h at 20°C so that -1 oocytes were ovulated and fertilized prior to confocal microscopy.For photoconversion of mKikGR, individual plates of one-day-old The diameter of LDs present in −1 oocyte and +1 embryos was manually fitted using the spot function in Imaris. The shortest distance between peri-LD cages and plasma membrane was computed by the spot and surface functions of Imaris XT.seip-1(tm4221) allele were backcrossed four times with wild-type males right before measurement. L4 larval P0 animals of each genotype were singled to individual plates and transferred to new plates every 24–48 h until egg-laying ceased. Hatched F1 animals were monitored and counted 24–48 h after the removal of the P0.Animals carrying the Four one-day-old adults  per replicate per strain were transferred to a new plate and allowed to lay eggs at 20°C. After 3 hours, adults were removed and the plate was stained with 500 μl 1 mM BODIPY in 1 × PBS for 3 h. The percentage of BODIPY-positive embryos was calculated using a fluorescence stereomicroscope. For comparison upon knockdown of different genes, the setup was similar as described above except that the age of adults varied in order to ensure both the efficiency and specificity of the knockdown. For constitutive knockdown, L4 larval P0 animals were plated and one-day-old F1 animals were used for scoring the percentage of BODIPY-positive embryos. For intestine-specific knockdown, L4 larval animals were plated and scored 48 h later. For germline-specific knockdown, L4 larval animals were plated and scored 24 h later.https://chopchop.rc.fas.harvard.edu/) and cloned into pDD162 (Addgene). The sgRNA/Cas9 vectors were injected into adult C. elegans along with the corresponding repair templates and co-injection markers. The transgenic P0s were singled and their progeny were selected with 5 mg/ml hygromycin B (InvivoGen). Viable F2s were further screened using a fluorescence stereomicroscope and PCR-based genotyping to verify knock-in alleles. For knocking out a target gene, 2-4 sgRNAs were injected along with co-injection markers. F1s from the transgenic P0s were singled and genotyped for deletion. For each knock-in/knock-out strain, at least two independent alleles were acquired and examined for consistency. All constructed alleles were backcrossed two times with wild-type males before further characterization.Tagging of the endogenous genes was performed as described previously . In brie


Mechanistically, the miR-144 directly targets the 3`-UTR of IRF5 and represses the expression of IRF5 in DCs. Ectopic expression of miR-144/451 by lentiviruses downregulates the levels of IRF5 and suppresses DCs function. In addition, knockdown of IRF5 by shRNA significantly inhibits activities of the miR-144/451-/- DCs. Expression of miR144/451 was decreased in DCs from both patients with IBD and mice with DSS-colitis compared with controls. Human PBMC derived DCs were downregulated expression of miR144/451 after LPS stimulation. In the DSS-induced colitis mice model, we showed that ablation of the miR-144/451 gene causes severe colitis, and their DCs from both periphery and MLN expressed higher co-stimulatory molecules and pro-inflammatory cytokines than wild-type mice. In addition, DCs isolated from miR-144/451-/- mice transfusion exacerbates mice colitis. In the bone marrow transplanted chimeric mice model, we show that miR-144/451-/- bone marrow transplantation deteriorated DSS-induced colitis. At last, we treat the mice with miR-144/451 delivered by chitosan nanoparticles revealing protective effects in DSS-induced colitis mice. Thus, our results reveal a novel miR144/451-IRF5 pathway in DCs that protects experimental colitis. The manipulation of miR-144/451 expression and DCs activation in IBD patients may be a novel therapeutic approach for the treatment of inflammatory diseases.The microRNAs miR-144/451 are highly conserved miRNA that is strongly induced during erythropoiesis. Despite the biological functions of miR-144/451 have been extensively studied in erythropoiesis and tumorigenesis, few studies have been conducted in immune responses. In this study, we showed that miR-144/451 Inflammatory bowel diseases (IBD), including two main clinical entities: Cohn’s disease (CD) and ulcerative colitis (UC) are characterized by both acute and chronic inflammation in the intestine. The etiology and pathogeny of IBD are largely unknown. Accumulating evidence have revealed that IBD is caused by dysregulated function of local immune cells such as dendritic cells (DCs), macrophages and T cells, and context cytokine production in the intestine , 2.MicroRNAs are endogenous short (~19 to 22 nucleotides) RNA molecules that negatively regulate gene expression in animals and plants by targeting mRNAs for degradation or translation inhibition . There imiR-144/451 that is strongly induced during erythropoiesis and facilitates erythrocyte maturation in zebrafish, mice and human  and c-Myc followed by activation of the downstream pathway in erythroid cells and B-lymphocytes . Together, those evidences indicate that miR-144/451 contributes to the regulation of immune responses. However, the molecular mechanisms remain largely unknown.miR-144 and miR-451 are two highly conserved miRNA encoded by a bicistronic gene locus nd human –12. GATAnd human . Our prephocytes . miR-144phocytes . A recenphocytes . In addiInterferon regulatory factor (IRF) family are transcription factor comprises nine members in human, which play a critical role in antiviral defense, immune response, cell growth regulation and apoptosis , 20. IRFIn the current study, we use miR-144/451 knockout mice, dextran sulfate sodium (DSS)-induced colitis model, and human IBD patient samples to demonstrate that miR-144/451 expression is decreased in DCs from IBD patients and DSS induced colitis compared with that from healthy donors. We also find that miR-144 targets 3`-UTR of IRF5 mRNA to deregulate DCs activation and ability to prime T cells. Elevated activation of DCs from miR-144/451 KO mice acerbates DSS-induced colitis. Thus, our data indicate that miR-144/451 may repress DCs activation in DSS-induced colitis by targeting IRF5.+) mice lacking a 388 base pair segment of genomic DNA containing both the miR-144 and miR-451 precursors have been described previously (+) mice were provided by the Comparative Medical Center of Yangzhou University . CD45.1+ WT (C57BL/6J background) mice were obtained from the Jackson Laboratory . All animals used in this study were 8-12 weeks old. 293T cells from American Type Culture Collection were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), streptomycin and penicillin.miR-144/451 knockout . The written informed consent was provided from each individual.420 base pairs of miR-144/451 genomic DNA were cloned and inserted into a pLVX-acGFP1 lentiviral expression vector. Lentiviruses were packaged with vector pLP1, pLP2 and pLP/VSVG in 293T cells by Lipofectamine 3000 Reagent (ThermoFisher). 293T cells or DCs were infected with lentiviruses together with polybrene (10 µg/ml) for 24 or 48 hours. The infection rate of lentivirus was tested in 293T cells as shown in http://rnaidesigner.thermofisher.com/rnaiexpress, ThermoFisher). shRNA1: 5`- GGGACAACACCATCTTCAAGGCTCGAGCCTTGAAGATGGTGTTGTCCCTTTTT-3`, shRNA2: 5`-GGTTGCTGCTGGAGATGTTCTCTCGAGAGAACATCTCCAGCAGCAACCTTTTT-3`. Target sequences and negative control NC-shRNA  were inserted in the pLVx-shRNA2 vector. Lentiviruses were packaged in 293T cells.IRF5-shRNA was designed using BLOCK-iT RNAi Designer (b (5041.16.1), I-A/I-E (M5/114.15.2), CD1d (1B1), TNF-α (MP6-XT22), IL-6 (MP5-20F3), CD3 (17A2), CD4 (GK1.5), CD8 (53-6.7), CD69 (H1.2F3), NKG2D (CX5), IFN-γ (XMG1.2) and anti-human-CD11b (M1/70), CD11c (3.9), CD80 (2D10.4), CD86 (IT2.2), TNF-α (MAb11) were obtained from BioLegend  or Thermo Fisher . Cells were treated or permeabilized, stained with corresponding antibodies, and analyzed by BD FACSVerse system and FlowJo software .Antibodies used in flow cytometry include anti-mouse-CD11b (M1/70), CD11c (N418), CD80 (16-10A1), CD86 (GL-1), H2K+ DCs , CD8+T (Ly-2 MicroBeads), and CD4+T (L3T4 MicroBeads) cells were sorted by magnetic activated cell sorting (MACS) according to the manufacturer’s protocol.CD11c−ΔΔCT method. Primers for microRNA quantification were designed by miRAN Design software : RT-miR-144-5`-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACTTAC-3`, miR-144F5`-GCGCGGGATATCATCATATACT-3`; RT-miR-451-5`-GTCGTATCCAGTGCA GGGTCCGAGGTATTCGCACTGGATACGACAACTCA-3`; miR-451F-5`-CGCGAAACCGTTACCATTAC -3`. miRNA 1st Strand cDNA Synthesis Kit by stem-loop and miRNA Universal SYBR qPCR Master Mix (Vazyme Biotech) were used for microRNA quantification.Cells with corresponding treatment were harvested and stored in TRizol reagent . Total RNA was extracted and assessed quality by NanoDrop (Thermo Fisher). RNA was reverse transcribed to cDNA using Takara Prime Script RT reagent kit with gDNA Eraser (Takara Bio Inc). The primer sequences used in this study were as follow, IRF5: foreword-5`-CCTCCCAACGCACCCTATT-3`, reverse -5`-ATCAGCAGGTCAGGCAAGA-3`. GAPDH: foreword-5`- CCACTCACGGCAAATTCAAC-3`, reverse -5`-CTCCACGACATACTCAGCAC-3`. The relative expression level was calculated using the 2Fresh isolated or treated DCs were rinsed with cold PBS and lysed in lysis buffer . The protein concentration was measured using NanoDrop. Equal amounts of protein were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes; nonspecific sites were blocked with 5% BSA in TBST and the membranes were then incubated with dilutions of the primary antibody as recommended by the manufacturer. The antibodies used are as follows: anti-IRF5, anti-β actin  , HRP conjugated anti-rabbit secondary antibody . Western blots were visualized using the enhanced chemiluminescent (ECL) reagent (Thermo Fisher).228 base pair segments of IRF5 3’-UTR containing the target sequences (wild type and mutated) of miR-144 were cloned into the pGL3-BS vector  in drinking water for 6 days followed by DSS-free water . The bodi.e., for 20 g mouse, administer 600 µg of busulfan at day -7, -5 and -3). Donor bone marrow cells were freshly isolation from femur and tibia of donor mice, and then adoptively transferred to recipient mice (2 × 106 bone marrow cells per mouse) via tail vein. Recipient mice were supplied with water containing gentamicin and erythromycin for 2 weeks before the followed procedures. For the DCs transplantation model, CD11c+ DCs were sorted and transferred to recipient mice (2 × 106 cells/mouse via tail vein).In the BMT model, recipient mice were pretreated with busulfan  were mounted on glass slides and stained with hematoxylin and eosin (H&E). Pathological evaluations were performed based on an earlier study .in vivo delivery study, mice were treated with nanoparticles daily .Chitosan nanoparticles were prepared as previously described . The solp<0.05, **p<0.01, ***p<0.001.All data were presented as the means ± SEM. Differences between two samples were analyzed using an unpaired, two-sided Student’s t-test. Multiple comparisons were performed using two-way ANOVA, and p values were adjusted using Tukey’s method. p values of less than or equal to 0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Software Prism 9. Statistical significance was indicated as *b, I-A/I-E, CD1d) was not dramatically changed in DCs from KO mice   were both elevated in miR-144/451 KO mice. Whereas, we did not see any changes in macrophages including frequency, expression of co-stimulatory factor and MHC molecular in miR-144/451 KO mice compared with that in WT mice (data not show).miR-144 have being significantly destroyed in many types of cancers including leukemia, gastrointestinal cancers, pancreatic cancer, hepatocellular carcinoma, lung cancer, and breast cancer , 35. miR6 (BCL6) , human f6 (BCL6) , nuclear6 (BCL6) , Smad1 (6 (BCL6) , taurine6 (BCL6)  and FMS-6 (BCL6) . Despite6 (BCL6)  and tumo6 (BCL6) , 35, few+ cells during intestinal inflammation .WG and ZL: the conception and design of the study. ZL, XX, MG, and QC: acquisition of data, analysis and interpretation of data. ZL: wrote the main manuscript text, prepared figures, statistical analysis. GL, XJ, WX, and JZ: methodology; WG and DY: Supervision. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China , the Natural Science Foundation of Jiangsu Province, China (Grant number: BK20180925); the “Six peaks” Talent Project of Jiangsu Province.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Scientific Reports 10.1038/s41598-022-15870-8, published online 08 July 2022Correction to: The original version of this Article contained an error in the grant number stated in the Acknowledgments section.“Project financially supported by Polish National Science Centre research Grant Preludium Bis No. UMO-2019/35/B/NZ8/01388.”now reads:“Project financially supported by Polish National Science Centre research Grant Preludium Bis No. UMO-2019/35/O/NZ7/00619.”The original Article has been corrected.


Decoy receptor 3 (DcR3) belongs to the tumor necrosis factor (TNF) receptor superfamily and neutralizes TNF ligands, including FasL and TRAIL, to prevent T activation during T-cell priming. However, the cellular mechanisms underlying acute cell-mediated rejection (ACMR) remain unknown.in vivo and in vitro. FasR RNA knockdown in immortalized CD4+CD8+ T-cells was used to survey the role of DcR3 on FasR/Fas-associated protein with death domain (FADD)/caspase 8 pathway and its cross-link to TNF receptor-associated factor 1 (TNFR1)-associated death domain protein (TRADD) in suppressing TNFR1. TNF/TRADD knockout mice were used to show the importance of TNF adaptor protein.We generated DcR3 transgenic (Tg) mice and mice with high DcR3 expression (HDE) to study both +CD69+, CD4+CD44+, and CD4+CD25+Foxp3+ when compared with isotype IgG1 and its co-treatment with FasL/TRAIL after exposing to bone marrow-derived dendritic cells (BMDCs) that carried alloantigen with male H-Y and minor antigenic determinant. Interleukin-17 and interferon-γ productions by BMDC-activated T-cells were lowered after co-treating with DcR3.Fc. DcR3.Fc induced effector T-cells (Teffs) and was susceptible to FasR-mediated apoptosis through the FADD/TRADD/caspase 8 pathway. After exposing to DcR3.Fc, TRADD was silenced, likely turning down the inflammatory response. The systemic effects of DcR3 Tg mice and HDE phenotype induced by the promoter of cytomegalovirus not only attenuated ACMR severity but also ameliorated the high serum creatinine and blood urea nitrogen levels even with high T-cell exposure frequencies. Besides this, DcR3 has minor biological effects on both MHC-matched and MHC-mismatched models.DcR3.Fc suppressed C57BL/6 female T-cell activation and transformation into CD4via interfering with TNF ligand-mediated reverse signaling and possibly promoting Teff apoptosis through FasR upregulation. Our findings supported that the decoy receptor is involved in T-cell modulation in kidney transplant rejection.High DcR3 doses protect renal tubular epithelial cells from acute T-cell attack during the T-cell priming stage  DcR3,henotype . DcR3 alhenotype ; howeverhenotype . Howevervia apoptosis induction both in vitro and in vivo , is positively correlated with both T-cell mediated rejection (TCMR) severity and adverse allograft survival in human renal transplant recipients (RTRs) . TherefoFor immunohistochemical (IHC) staining, monoclonal antibodies (mAbs) were used against cleaved caspase 3, which was followed by the same procedure with LIGHT and CTLA4. Anti-FasL, anti-LIGHT, anti-TRAIL, and anti-TL1A antibodies were used for western blot. The antibodies used for immunofluorescence (IF) double staining were anti-CD45, CD3, CD4, and CD8 Ab for T-cell markers, anti-CD20 Ab for B-cell marker, and anti-CD68 Ab for macrophage. For DcR3 Ab, the incubation duration was overnight at 4°C. Immunoreactivity was detected with fluorochrome/secondary antibodies, including anti-goat  and anti-rabbit  IgG for DcR3 and CD markers, respectively. We used 4′-6-diamidino-2-phenylindole to bind with double-strand DNA in the nucleus, aided under 358 nm (blue light). An imaging technique was used to increase the optical resolution and contrast of micrographs from confocal microscopy . The reagents for T-cell study included FasL (Abcam), TRAIL , DcR3.Fc , and human IgG1 alone (Millipore). The mAbs for flow cytometry included CD4, CD8, CD25, Foxp3, CD44, CD69, FasL, FasR, TRAIL-R2, TNFR1, and TNFR2  was used to stimulate the BMDCs for 24 h. Initially, RBC was lysed using the ACK lysing buffer , and the presence of mature BMDC was confirmed using positive staining with MHCII (eBioscience) and CD11c-PE (eBioscience) (Bone marrow-derived dendritic cells (BMDCs) from 5–7-week-old male C57BL/6 mice were cultured in a petri dish with 6 ml Roswell Park Memorial Institute (RPMI) and 20 ng/ml granulocyte–macrophage colony-stimulating factor . RPMI and GM-CSF were changed on days 3, 6, and 8 while culturing BMDCs. On day 9, 100 ng/ml lipopolysaccharide . Fluorescence-activated cell sorter-sorted CD4+/CD8+ T-cells were obtained from female C57BL/6 lymph nodes (LNs) and spleen : wild-type (WT) mice, DcR3 Tg mice, WT mice with empty vector, and WT mice with hDcR3 plasmid injection  mice, B6 TNF/TNF receptor-associated factor 1 (TNFR1)-associated death domain protein (TRADD) double knockout (DKO) mice, and B6 GFP knock-in mice supplied by Dr. NJ Chen . The animals were maintained on a standard mouse chow diet, adequate room temperature, humidity, and light/dark cycle. The B6 DcR3 Tg mice were characterized by a 4.7-kb CD68-DcR3 cassette annealed to the CD68 promoter , and DcRin vitro ACR co-culture system included naïve or activated primary T-cells from female mice and RTECs from male mice together with male BMDCs, GM-CSF, and rIL-2, and those distinct patterns in Teff and Treg cells upon activation with TCR co-stimulation.The 6 activated CD4+/CD8+ female T-cells with amplified H-Y memory  vector  with EndotoxinOUT™ Resin . The expression constructs or the empty vector was administered to the B6 WT mice through the tail vein via hydrodynamics-based gene delivery using a single dose of 50 μg/10 g body hDcR3 plasmid (n = 17) (The human DcR3 (hDcR3) plasmid was prepared from (n = 17) . The anihigh) or low (pCMV-hDcR3low) DcR3 expression phenotype regulated the disease. High and low DcR3 expression was defined by serum DcR3 protein levels above and below the median level (37.0 ng/ml), respectively. The interquartile range was 10.5–66.5 ng/ml.WT mice transfected with hDcR3 plasmid with high  with or without 100 ng/ml FasL (+CD8+ T-cells (MOHITO) (Primary CD4/ml FasL . The oth/ml FasL . For flo(MOHITO)  with Bal+/CD8+ T-cells were incubated with CFSE and then co-cultured with male BMDCs, followed by addition of various reagents for 36 h. Proliferation was evaluated by flow cytometry with CFSE dye incorporation.To determine the effects of DcR3 on T-cell priming, female CD4in situ cell death detection kit (Vazyme) was used according to the manufacturer’s instructions. For each sample, TUNEL-positive tubular or T-cells were counted in 10 non-overlapping fields. Before and after DcR3.Fc treatment, the cells were double-stained with annexin-V-FITC and propidium iodide .For the transferase dUTP nick-end labeling (TUNEL) assay, an 2-wide pieces and digested with 100 μl collagenase. After digestion, the supernatant was sieved through two nylon meshes . Only the 75–80-μm sieves yielded a large number of proximal tubules. A 2-ml suspension containing Percoll 45% (Sigma-Aldrich) and a cell suspension in 1:1 proportion were centrifuged at 4°C and 13,000 × g for 30 min. The bottom layer was then retrieved  was used to wash the harvested kidneys. Then, the native renal capsule and renal hilum were removed, and the renal cortices were dissected visually into approximately 1- to 2-mmetrieved .6–5 × 106 T-cells, and 6-well, 60-mm, or 10-cm culture dish).The endogenous protein was knocked down by transiently transfecting the cell lines with shRNA oligonucleotides. FasR shRNA (shFasR) and pLKO_TRC001 control  were electroporated into MOHITO cells using the Neon™ Electroporation Transfection system -conjugated  antibodies against target proteins and mouse HRP-conjugated  antibodies against beta-actin and GAPDH. Finally, the blot was subjected to chemiluminescent detection according to the manufacturer’s instructions. The gel analysis and quantitative values were obtained using Gene Tools .For real-time polymerase chain reaction (qPCR), the total RNA (cells and kidney lysates) was purified using the TRIzol reagent according to the manufacturer’s instructions. The qPCR analyses were carried out using StepOnePlus™ Real-Time PCR System (ABI) and Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) as a qPCR reagent kit. The analyses were carried out using a Lightcycler system (Roche), with GAPDH RNA serving as an internal control. The qPCRs were performed to detect RNA expressions in mice and humans (in vitro and in vivo results were expressed as the mean ± standard deviation (SD) or standard error (SE). The results from multiple groups were analyzed using one-way analysis of variance (ANOVA). We used Kruskal–Wallis test followed by Bonferroni post-hoc analysis for multiple testing. For two independent groups, nonparametric Mann–Whitney U-test was used. The percentages of infiltration in gross view of mouse kidneys were quantified with the software Image-Pro Plus 6.0 . Analysis of the 3,3′-diaminobenzidine-positive area was carried out using Image J (version 1.16). Dot plots and graphs were generated with the software GraphPad v5. Receiver operating characteristic curves were compared using the MedCalc software .The +/CD8+-induced inflammatory response  and time dependent. Such changes gradually diminished in DcR3 Tg mice (high) or low-expression (pCMV-hDcR3low) phenotype regulated the disease after a high-dose (3 × 106) T-cell injection, reducing tissue infiltration  and DcR3 in areas proximal to the plasma membrane of inflamed cells of ACR DcR3 Tg mice. A similar CD68 and DcR3 co-localization was found in ACR DcR3 Tg mice after CD4On day 7 after ACR, the FasL, TRAIL, and LIGHT mRNA expressions in DcR3 Tg mice were significantly suppressed. Moreover, the pro-inflammatory cytokines  were downregulated and the anti-inflammatory cytokines  were upregulated in DcR3 Tg mice than those in WT mice  or activated T-cells . As expected, we potentially found protective effects of DcR3 on RTECs after adding DcR3.Fc alone or DcR3.Fc with FasL or TRAIL using the TUNEL assay  than tho+/CD8+ T-cells were incubated with activated male BMDCs under different reagents for 24 h, and the T-cell phenotype was examined. DcR3 was critical for CD4+CD69hi, CD4+CD44hi  and TRAIL + IgG1  but are less induced with FasL + DcR3.Fc  and TRAIL + DcR3.Fc   than that with IgG1 control (7.4 ± 1.6%) or with TRAIL + DcR3.Fc (2.5 ± 1.2%) than that with TRAIL + IgG1 (8.5 ± 3.9%) , pro-caspase 8, active caspase-8, and TRADD in protein complex  formation in vivo, we used the other ACR TNF/TRADD DKO mice (To determine whether DcR3 promotes TNFR2 expression n = 12) and with biopsy-proven ACR   suppressed T-cell priming through FasL/TRAIL-mediated reverse signaling , DcR3 promoted Teff apoptosis through FasR/FADD/caspase 8 upregulation prone to anti-Th1 phenotype  has suggested that the C-terminal death domain of FADD (80–205) inhibits TNF-induced apoptosis without inhibiting TNF-mediated NF-κB activation . Moreove+ MOHITO cells appeared to follow TNF/TRADD DKO distribution after comparing it with WT murine T-cells. Interestingly, the kidney histology of ACR WT mice concurrently injected with pCMV-hDcR3 via the tail vein showed less destruction than that of TNF/TRADD DKO mice. However, the absence of TNF/TRADD signaling pathway in recipients with the alternative TNFR2 phenotype limits alloreactivity in vivo . The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Institutional Animal Care and Use Committee (IACUC) of the National Yang-Ming University (IACUC1001106) and Taichung Veterans General Hospital , Taiwan.S-CW, N-JC, and D-CT conceived and designed the experiments. S-CW, M-CW, N-JC, S-LH, and D-CT performed the experiments. S-LH provided the DcR3 Tg mice. S-CW, M-CW, N-JC, and D-CT analyzed the data. S-CW, N-JC, and D-CT wrote the paper. All authors contributed to the article and approved the submitted version.We are deeply indebted to Taichung Veterans General Hospital, Taichung, for providing the grants for this study . This study was also supported by the Taiwan Ministry of Science and Technology (MOST 106-2314-B-075A-003 and MOST 109-2314-B-075-097-MY3) and Foundation for Poison Control (2022-04).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Human tuberous sclerosis (TSC) is mainly caused by genetic mutations of tuberous TSC1or TSC2. Recent studies found that TSC1 deficiency promoted classical M1 macrophage polarization. However, whether TSC1 regulates other inflammatory cytokine expression in lipopolysaccharidem (LPS)-stimulated macrophages is unknown. Herein, we studied the cytokine expression profile of wild-type (WT) and TSC1-deleted macrophages after LPS stimulation in vitro and the pathogenesis of dextran sodium sulfate (DSS)-induced colitis in mice with myeloid-specific TSC1 deletion (TSC1cKO mice). We found that TSC1-deficient macrophages exhibited the enhanced secretion of interleukin-17A (IL-17A), IL-17F, and interferon-gamma (IFN-γ) in response to LPS stimulation in vitro. This is in contrast to LPS-stimulated WT macrophages, which usually do not. Importantly, TSC1cKO mice exhibited exacerbated DSS-induced acute colitis with severer symptoms. MTOR deletion or rapamycin treatment significantly reversed the enhanced expressions of IL-17A, IL-17F, and IFN-γ in LPS-stimulated TSC1-deficient macrophages in vitro and rescued the enhanced DSS-induced colitis in TSC1cKO mice, indicating that TSC1 deficiency increased these cytokine productions in an mTOR-dependent manner. RNA-sequencing and molecular studies indicated that TSC1 deficiency enhanced the aerobic glycolysis process and the activities of mTOR-STAT3-RORγT pathway in LPS-stimulated macrophages. Inhibition of aerobic glycolysis, STAT3, or RORγT reversed IL-17 and IFN-γ expression in LPS-treated TSC1-deficient macrophages. Thus, TSC1 is essential for macrophages to shut down IL-17A, IL-17F, and IFN-γ expression during LPS stimulation by suppressing the aerobic glycolysis process and mTOR-STAT3, RORγT, and T-bet pathways. The present study uncovered the key role of TSC1 in shutting down IL-17A, IL-17F, and IFN-γ expressions in LPS-treated macrophages. The genetic mutation of tuberous sclerosis complex 1 (TSC1) or tuberous sclerosis complex 2 (TSC2) genes causes tuberous sclerosis  disorder in humans, presenting with multi-organ hamartomas, renal cysts, cortical tubers, hypomelanotic macules, facial angiofibroma, progressive splenic hamartomas, subependymal giant cell astrocytoma, and drug-resistance. TSC1 anin vivo and in vitro [Macrophages are important components of the immune system, responsible for host defense, pathogen limitation, apoptotic cell clearing, and tissue repair , 15. Thein vitro , 17. A uin vitro . Thus, tin vitro. Molecular and biochemical mechanism studies suggested that the enhanced expression levels of IL-17A, IL-17F, and IFN-γ in TSC1-deleted macrophages are mTOR-glycolysis-dependent. Mice with a myeloid-specific deletion of TSC1 gene (TSC1cKO) displayed severe dextran sodium sulfate (DSS)-induced colitis accompanied by high levels of IL-17A, IL-17F, and IFN-γ. Our present study uncovered the essential role of TSC1-mTOR in turnoff of Th17 cytokine expression in macrophages and ameliorating acute colonic inflammation via metabolism-dependent pathways.Recent studies have highlighted the importance of the TSC-mTOR signaling pathway in the regulation of macrophage activation, contributing to various inflammatory diseases, including colitis and sepsis , 19, 20.The monoclonal antibodies (mAbs) for flow cytometry analysis used in this study are listed: anti-mCD11b-BV510 , anti-mCD45-BUV395 , anti-mCD45-PE-Cy5 , anti-mLy6G-FITC ,anti-mF4/80-PE-Cy5 , anti-mF4/80-PE , anti-mIL-17A-PE , anti-mIL-17A-BV421 , anti-mIFN-γ-PE, anti-mIFN-γ-FITC , anti-m CD16/CD32 . There are antibodies used for western blot assay: anti-β-Actin , phospho-Stat3(Tyr705) , phospho-Stat3 (Ser727) , ROR gamma (t) , phospho-S6 (Ser235/236) , phospho-eIF4E (Ser209) , eIF4E (C46H6) , anti-rabbit secondary antibodies , prestained protein ladder . Other major reagents used in this study: Recombinant murine M-CSF , LPS . All reagents were used following the manufacturers’ instructions.loxp/loxp; TSC1cKO) were generated by crossbreeding TSC1loxp/loxp mice with LysM-Cre mice expressing Cre recombinase under the control of Lysozyme promoter (LysM-Cre) [loxp/loxp/mTORloxp/loxp; TSC1/mTORcKO) in myeloid cells, were obtained from crossbreeding TSC1loxp/loxp and mTORloxp/loxp mice. The 4-5-week-old C57BL/6J mice and 6-8-week-old female CD45.1 C57BL/6J mice were purchased from Beijing University Experimental Animal Center . The TSC1loxp/loxp mice were gifts from Dr. Hongbin Zhang . The LysM-Cre mice were kindly offered by Dr. Lianfeng Zhang . All mice were bred and maintained in the specific-pathogen-free animal facility of the Animal Experiment Centre of the Institute of Zoology in Beijing, China. All experiments were ethically performed according to the Institute of Zoology’s Institutional Guidelines for the Care and Use of Laboratory Animals. The flowchart and procedures of the in vitro and in vivo experiments were shown in Mice with TSC1 deletion in their myeloid cells (LysM-CreTsc1ysM-Cre) , 23. The2 to induce bone marrow-derived macrophages (BMDM) [2 for 2 h [2 with stimulations for an indicated amount of time.The euthanasia method for mice used in this study is carbon dioxide asphyxiation which mice were placed in a new cage and then euthanized by the air of 100% carbon dioxide inhalation . The bons (BMDM) , 21, 25. for 2 h . The purColitis was induced in WT and TSC1cKO mice via 2.5% DSS  in drinking water for 6 days , 28. Eac2 to obtain adherent colonic macrophages.Colonic macrophages were collected from the mice colons by enzymatic digestion based on the established method . The colH&E staining was performed on the basis of the protocol from previous studies , 32. In The 4-5-week-old TSC1cKO and WT mice were injected intraperitoneally with Rapa (50 μg/kg body weight) or carboxymethyl cellulose sodium (CMC) for 3 days.+F4/80+ macrophages, PEMs treated in vitro were plated in 24-well plates at a density of 1 X 106/ml and incubated for 6 h at 37 °C with 5% CO2. Single-cell suspensions were prepared and then washed once using cold PBS blocked with anti-CD32/CD16 to avoid nonspecific binding [To analyze the production of IL-17A and IFN-γ by CD11b binding . They weCell culture supernatants were examined for IL-17A with Mouse IL-17A ELISA Kits  according to the manufacturer’s instructions . The 96--ΔΔCt method.Total mRNA was extracted using Trizol reagent  or a MicroElute Total RNA kit  according to the manufacturers’ instructions . RNA quaP-value < 0.05 and |Foldchange| > 2. We applied Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY [www.genome.jp/kegg) and KEGG Orthology-Based Annotation System (KOBAS) 3.0 [http://kobas.cbi.pku.edu.cn/) to perform pathway analysis. In addition, gene set enrichment analysis (GSEA) was carried out using the REACTOME database [https://string-db.org/) and visualized by Cytoscope [We prepared the total mRNA as described above and followed the reported method for data processing , 38. Pro PATHWAY  (availabBAS) 3.0  (availabdatabase . The anadatabase  (availabytoscope .6 cells/well) seeded in a 24-well plate were incubated for 2 days in vitro. Supernatants of cell culture medium were collected, and then the glucose and lactate levels were measured using the 1450 MicroBeta JET (Perkin Elmer) [Glucose and lactate levels were immediately determined after macrophages (2 x 10n Elmer) . Glucose6 cells/well) were cultured in DMEM medium with 5% FCS in 24-well plates. The cells were treated with indicated stimulus, LPS (100 ng/ml) or Rapamycin (100 nM) or LPS and Rapamycin, for the indicated time. After stimulation, the cells were washed twice with cold PBS and then lysed in RIPA buffer  with protease and phosphatase inhibitor cocktails  for 10 mins on a rocker at 4 °C. The protein concentration was determined via BCA assay. The protein contents were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes . Each polyvinylidene fluoride membrane was blocked with TBST  in 5% skim milk for 1 hour at room temperature, then incubated with primary antibodies overnight on a shaker at 4 °C. Subsequently, HRP-coupled secondary antibody was added, and protein samples were detected by chemiluminescence . Anti-β-actin mAb (working concentration: 1:10000) determined in previous studies was chosen as a protein loading control [Macrophages cells  or mean ± standard error of the mean (SEM) as appropriate, which is representative of at least 3 independent experiments with a minimum of 3 biological replicates. Log-rank tests were used for mouse survival assays. Kolmogorov-Smirnov and F-value tests were performed to analyze normal distribution and homogeneity of variance of data in order to determine normality of data. Student’s unpaired two-tailed t-test for comparison between two groups, or one-way ANOVA analysis for multiple groups were used as paramedic methods. Mann-Whitney U test was used as the none-paramedic method. loxp/loxp mice with transgenic mice that carried lysozyme (LysM) proximal promoter-mediated Cre recombinase . Then we detected the mRNA expression levels of a set of cytokines in freshly isolated wild type (WT) and TSC1cKO mouse peritoneal resident macrophages after LPS stimulation for 6 h by real-time PCR. The LPS treatment induced significantly higher tumor necrosis factor-α (TNF-α), interleukin 12p40 (IL-12p40), and interleukin 23 (IL-23) mRNA expression in freshly isolated TSC1cKO peritoneal macrophages compared with WT macrophages  or with TSC1 and mTOR double deletion  in myeloid cells. Peritoneal macrophages isolated from TSC1/mTORcKO mice expressed almost undetectable levels of IL-17A, IL-17F, and IFN-γ, the same as the WT and mTOR deletion cells after LPS stimulation as determined by real-time PCR  on LPS-stimulated WT and TSC1cKO macrophages. Through differential gene analysis, we found that compared with WT macrophages, TSC1 deficiency in macrophages led to the upregulation of 2,451 genes and downregulation of 1,651 genes under LPS stimulation. Among them, the upregulated genes were mainly enriched in cytokine interaction pathways , macrophage pattern recognition receptor-related pathways NOD-like Receptor (NLR) signaling pathway and TLR-like Receptor (TLR) signaling pathway), inflammatory signaling pathways , and metabolism-related signaling pathways  . The aboRapa treatment or mTOR deletion decreased the enhanced levels of STAT3 activation caused by TSC1 deficiency in macrophages, as indicated by the p-STAT3 levels found by western blot analysis . Rapa trIt has been proposed that mTORC1-dependent metabolic reprogramming of aerobic glycolysis is especially important for Th17 cell development -54. GlucMacrophages play a vital role in controlling the duration and magnitude of host defense against pathogenic infections and inflammatory responses . In geneGenetic analysis illustrated that the cytokine interaction pathway and inflammation-associated signaling pathway were enriched in TSC1-deficient macrophages under LPS stimulation. STAT3 and its related signaling pathway were significantly upregulated in LPS-treated TSC1-deficient macrophages. It has been reported that mTOR played a crucial role in inducing Th17 differentiation by promoting STAT3 activation -63. ActiIn addition, IFN-γ is usually produced by NK, Th1 cells, and other immune cells. Monocytes/macrophages could express IFN-γ after IL-12/IL-18, LPS/ATP, and IL-23 stimulation, respectively , 66. LPSIn conclusion, we herein demonstrated that TSC1-mTOR signaling pathway negatively regulated Th1/Th17-type cytokine expression in LPS-stimulated macrophages and exacerbated pathogenesis of colitis in mice via reprogramming metabolism and STAT3-RORγT/T-bet pathway. The identification of the master role of TSC1 in silencing Th1/Th17-type cytokine expression in LPS-stimulated macrophages provided a new molecular mechanism for the unclassical polarization of macrophages and might offer novel approaches to prevent macrophage hyperactivation-related diseases.


Despite COVID-19 pandemic significantly impacting mental health, few studies evaluated effects on perinatal mental health.Therefore, we aimed at assessing pregnant and puerperal women during first and second COVID-19 waves.70 women  consecutively afferent to our outpatient service for Perinatal Mental Health (March 2020-March 2021) were administered Edinburgh Postnatal Depression Scale (EPDS), Fear of COVID-19 (FCV-19-S), Coronavirus Anxiety Scale (CAS) and Wijma Delivery Expectancy/Experience questionnaire (WDEQ).Women who reported last menstruation date (LMD) in 2019 second semester showed higher EPDS scores (p=0.026), those with estimated delivery date (EDD) in 2021 second semester showed higher CAS scores than those with EDD in 2020 first semester (p=0.020) or in 2021 first semester (p<0.001). Women with clinically significant EPDS Scores reported higher FCV-S-19 (p=0.005) and CAS (p=0.003). Subjects with a previous psychiatric hospitalization showed higher FCV-S-19 (p=0.003). A weak positive correlation  has been observed between FCV-S-19 and EPDS. Furthermore, there was a strong positive correlation  between CAS and EPDS and between CAS and FCV-S-19 . All subjects showed high scores for tocophobia after experiencing delivery.COVID-19 pandemic significantly impacted pregnant and/or postpartum women also without a previous psychiatric condition. Early identification and screening tools should be routinely provided to all pregnant and postpartum women.No significant relationships.