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: Decreased inhibitory effect on type I IFN response stimulated by IFN-α/β in rD1 EV-A71-infected cells. (A) Induction of ISGs in rD1-infected cells. HeLa cells were infected with rFY or rD1 EV-A71 for 24 h, followed by treatment with or without IFN-α (25 ng/μL) for another 2 h. Total RNA was prepared from the cells for real-time RT-PCR to measure relative levels of the ISG gene transcripts. Values represented mean ± SD from triplicates of independent experiments. t tests were conducted on values from different groups. *P<0.05; **P<0.01; ***P<0.001. (B,C) Decreased reduction of ISGs and KPNA1 in rD1-infected cells. HeLa cells were infected with rFY or rD1 EV-A71 for 24 h, followed by treatment with or without IFN-α (B) or IFN-β (C) (25 ng/μL) for another 2 h. Cell lysates were prepared for western blot analyses with antibodies for MX1, OAS1, or KPNA1. (D) Increased translocation of p-STAT1/2 into the nucleus in rD1-infected cells. HeLa cells were infected with rFY or rD1 EV-A71 for 24 h, followed by stimulation with IFN-α (25 ng/μL) for another 30 min. The cytosolic and nuclear fractions were prepared from the cells for western blot analyses with antibodies for p-STAT1 or p-STAT2. β-tubulin and histone H3 were detected as loading controls for cytosolic and nuclear fractions, respectively.

Fig 4: Regulation of viral replication and oncolysis by IFN-regulatory factor 9 (IRF9). Cells were transfected with either control or IRF9-specific siRNA #2 followed by infection with AdGFP or OAdmCherry at a multiplicity of infection (MOI) concentration of five alone or in combination with 500 nM palbociclib for 48 h. (A) Type I IFN signaling and expression of E1A viral protein was assessed by immunoblotting with the indicated antibodies. Quantitative densitometry analysis is shown relative to loading control normalized to palbociclib-sensitive control-treated cells (except for MxA in MDA-MB-231 cells and E1A blots where samples were normalized to OAdmCherry Ctrl). (B) mCherry expression was evaluated by fluorescence microscopy. Bright field images illustrate cytopathic effect (CPE). Scale: 200 µm. Images are representative of three independent experiments. (C) Cell lysates were harvested and combined with collected supernatants to determine OAdmCherry viral titers. (D) Cell number was assessed by crystal violet staining and calculated by measuring the absorbance of solubilized dye at 590 nm. Results are expressed as percent (%) of AdGFP vehicle-treated cells. Error bars, ±SEM of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; compared to the control unless otherwise indicated.

Fig 5: The CD133low population displays an interferon response, which can be suppressed by exogenous CD133.A673 cells (A) and NCH-EWS-1 cells (B) were incubated with anti-CD133 (AC133) antibody and were sorted into the CD133high and CD133low populations. Part of the CD133low population was infected with CD133-expressing lentivirus and selected with puromycin. The expression of IFNB1, Mx1, OAS1, p21, NELL2, and CD133 was analyzed by qRT-PCR (left) and immunoblotting (right). *p < 0.05 compared with the CD133high population and with the CD133low population expressing exogenous CD133.