Fig 2: EZH2 suppresses ERVs, LINE-1, and an interferon response downstream of NELL2 signaling.(A) Extracellular NELL2 signals to maintain histone H3K27me3 modification in ERVs and LINE-1 in Ewing sarcoma cells. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with 100 ng/ml of recombinant NELL2 protein for 24 hours. Histone H3K27me3 modification in ERVs and LINE-1 was analyzed by chromatin immunoprecipitation. *p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. (B) EZH2 expression is regulated by NELL2 signaling in Ewing sarcoma. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with the indicated concentration of recombinant NELL2 protein for 24 hours. The expression of EZH2 was analyzed by qRT-PCR. NELL2 silencing resulted in reduced EZH2 expression, which was restored by recombinant NELL2. *p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. (C) The CD133low population displays low EZH2 expression, which was rescued by exogenous CD133. Cell populations in Figure 2 were analyzed for the expression of EZH2 by qRT-PCR. *p < 0.05 compared with the CD133high population and with the CD133low population expressing exogenous CD133. (D) NELL2 signaling maintains EZH2 binding to ERVs and LINE-1. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool and were left untreated or treated with 100 ng/ml of recombinant NELL2 protein for 24 hours. The binding of EZH2 to ERVs and LINE-1 was assessed by chromatin immunoprecipitation. NELL2 silencing reduced EZH2 binding to ERVs and LINE-1, which was restored by recombinant NELL2. *p < 0.05 compared with control siRNA transfected cells and with cells transfected with NELL2 siRNA and treated with recombinant NELL2. (E) EZH2 suppresses an interferon response induced by NELL2 silencing. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool, followed by transfection of EZH2 or empty vector. The expression of the indicated genes was analyzed by qRT-PCR. *p < 0.05 compared with cells transfected with control siRNA and empty vector and with cells transfected with EZH2. (F) EZH2 suppresses growth arrest induced by NELL2 silencing. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool, followed by transfection of EZH2 or empty vector. Cell proliferation was assessed using the IncuCyte live-cell imaging system. (G) EZH2 expression does not affect siRNA-mediated NELL2 silencing. A673 cells were transfected with NELL2 siRNA pool or control siRNA pool, followed by transfection of EZH2 or empty vector. The protein levels of NELL2, IFNB1, Mx1, OAS1, and p21 were assessed by immunoblotting. Tubulin serves as a loading control.

Fig 3: Summary diagram describes that TINCR regulates breast cancer cell proliferation.TINCR-STAU1-OAS1 axis in breast cancer.

Fig 4: TINCR undermine the stability of OAS1 through binding to STAU1.A Mean centered, hierarchical clustering of transcripts altered in breast cancer cells treated with scrambled siRNA or si-TINCR 2#, with three repeats. B, C qRT-PCR analysis was used to validate the changes of several top regulated mRNAs upon TINCR depletion and overexpression in T47D and MCF-7 cells. D OAS1 mRNA expression upon STAU1 depletion, as detected by quantitative reverse transcription (qRT–PCR). E OAS1 mRNA expression was detected by qRT–PCR in pcDNA-TINCR and si-STAU1 co-transfected T47D and MCF-7 cells. F Protein level of OAS1 was detected by western blot assays in pcDNA-TINCR and si-STAU1 co-transfected T47D and MCF-7 cells. G Bioinformatics tool (http://bioinfo.bjmu.edu.cn/lncpro/) was used to evaluate the combination possibility between several top upregulated mRNAs and STAU1. Predictions with probabilities >0.5 were considered “positive”, indicating that the corresponding RNA and protein are likely to interact. OAS1 showed high possibility to interact with STAU1. H RIP assay was performed with IgG or STAU1 antibodies in T47D cells, and the coprecipitated RNA was subjected to qRT-PCR for OAS1. I The predicted TINCR binding site in OAS1 (OAS1-3′UTR-Wt) and the designed mutant sequence (YY2-30 UTR-Mut) are shown (up). Luciferase reporter gene assays of T47D and MCF-7 cells are shown (down) (OAS1-3′UTR-Wt + TINCR-NC group is seen as control). J, K TINCR and STAU1 control OAS1 mRNA stability. RNA stability assays were performed in T47D cells using Actinomycin D to disrupt RNA synthesis, and the degradation rates of the OAS1 mRNA were measured over 9 h. Values are shown as the mean ± s.d in three independent experiments. The mean values of two groups were compared via paired, two-tailed Student t tests.*P < 0.05; **P < 0.01.

Fig 5: OAS1 is a potent SARS-CoV-2 restriction factor.(A) OAS family genes in inducible CRISPRa ISG screen results. For A549-SunTag ACE2 and A549?STAT1-SunTag ACE2 screens, ISGs were ranked by SARS-CoV-2:Dox interaction adjusted p value (x axis). ß scores for SARS-CoV-2 infection status coefficient (DoxOn SARS-CoV-2 infected vs DoxOn mock infected) are plotted on the y axis, with points passing significance filters highlighted in red/blue for “antiviral”/“proviral” effects, respectively. OAS family members are labeled as indicated. (B) Images of A549-SunTag ACE2 and A549?STAT1-SunTag ACE2 cultures transduced with OAS1 gRNA or NTG, treated/not treated with Dox and infected as indicated with SARS-CoV-2 (M.O.I. = 2, 72 hours infection). Cultures were fixed and stained with Crystal violet to visualize cell viability. (C) SARS-CoV-2 growth curves measured by plaque assay. A549?STAT1-SunTag ACE2 cells expressing OAS1 gRNA or NTG gRNA were infected with SARS-CoV-2 (M.O.I. = 0.1) and culture media was collected at indicated time points (x-axis, hours post infection, hpi). Titer was determined by plaque assay on Vero-E6 cells. Statistical significance at each timepoint determined by two-sided Student’s t-test, *** p < 0.0005, ** p < 0.005, * p < 0.05. (D) Immunoblot analysis of OAS1 isoforms, OAS1p46 catalytic inactive mutant, OAS2, OAS3 and OASL. Indicated genes were expressed from lentivirus vectors and gene expression was induced by Dox treatment for 48 hours prior to immunoblotting with indicated antibodies. fLUC-expressing vector was used as a negative control. (E) Representative flow cytometry histograms for SARS-CoV-2 N protein in A549 ACE2 and A549?STAT1 ACE2 transduced with expression constructs for indicated OAS family member cDNAs, treated (red) or not treated (gray) with Dox, at 24 hours post-infection with SARS-CoV-2 (M.O.I. = 2). (F) Percent of infected (SARS-CoV-2 N protein positive) cells quantified across biological replicates for experiments described in (E). Values denote percent of infected cells in Doxon cultures relative to paired Doxoff cultures. Points represent individual biological replicates, black lines indicate mean values of biological replicates for each indicated cDNA. Red points indicate statistical significance (p < 0.05) as determined by paired ratio Student’s t-test.