Fig 2: 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 3: Interferon-stimulated genes (ISG) are regulated by EBV miRNAs. (A) 293T cells were cotransfected with individual EBV miRNA expression vectors or an empty vector as control and a reporter plasmid (p6898) containing a luc2 gene under the control of a chimeric promoter consisting of the ISG Mx2 promoter and five canonical ISREs. After 24 h, cells were treated with type I IFN and luciferase signals were measured the next day. As a control, an expression vector encoding human miRNAs (miR-373) was used (p6481). A further control was the IE1 gene from CMV, which is known to inhibit type I IFN signaling (49). P values were calculated by one-way ANOVA test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (B to G) 293T cells were cotransfected with the indicated wild-type luciferase reporter plasmids (WT) for CBP, IRF9, JAK1, JAK2, MAPK2, and IKKβ or reporter plasmids in which the predicted seed sequences were mutated (mut) together with or without an miRNA-encoding plasmid. The luciferase expression in these cells was assessed and normalized to lysates from cells cotransfected with the wild-type 3′-UTR reporter and empty plasmid (Ø). P values were calculated using the one-way ANOVA test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (H) Human primary B lymphocytes were infected with wild-type EBV (r_wt/B95.8) or EBV devoid of its miRNAs (r_ΔmiR). Five days postinfection, the transformed EBV-infected cells were counted and seeded at the same density. Four days later, the cells were lysed and the lysates were subjected to quantitative Western blot analysis using the Western blot stain-free TGX Bio-Rad normalization approach (Bio-Rad) and antibodies against IPO7, IFIT1, IRF9, and CBP. The applied protein amounts were analyzed and used to normalize the levels of the specific protein signals. The ratios of protein levels of cells infected with wild-type virus versus cells infected with mutant virus are shown. The figure summarizes the results of four independent replicates. (I) HEK293T cells were cotransfected with a pLCE-based EBV miRNA expression vector, a SIS-inducible element (SIE) responsive firefly luciferase reporter, and a pRL-SV40 renilla luciferase reporter as an internal control. Forty-eight hours after transfection, cells were either mock treated or treated with 10 U per ml universal type I interferon (uIFN) for 6 h and analyzed for luciferase activity. Values are normalized to mock-treated cells and reported relative to the pLCE control. Shown are the averages from three to four independent experiments performed in triplicate. Data were analyzed using Student's t test. *, P < 0.05; **, P < 0.02.

Fig 4: U-ISGs unresponsiveness depends on STAT1, STAT2 and IRF9 in Huh-7-SR cells. (A) Huh-7-SR cells were transfected with si-control, si-STAT1, si-STAT2, and si-IRF9. Then, 48 h after transfection, cells were harvested and immunoblotting of STAT1, STAT2 and IRF9 was performed. (B) mRNA levels of U-ISGs were measured by reverse transcription-quantitative PCR. (C) After transfection, Huh-7-SR cells were treated with an increasing dose of sorafenib for 24 h. **P<0.01 vs. siControl. IRF, interferon regulatory factor; si, small interfering; OAS1; oligoadenylate synthetase 1; IFI27, Interferon Alpha Inducible Protein 27.

Fig 5: Increased IRF9 expression in sorafenib-resistant liver cancer cells. (A) Procedure for establishing sorafenib-resistant liver cancer cells. (B) liver cancer cells were treated with an increasing dose of sorafenib for 24 h. Cell viability was measured by MTT assay. (C) Protein levels of STAT1, STAT2 and IRF9 from immunoblotting. (D) mRNA levels of STAT1, STAT2 and IRF9 from reverse transcription-quantitative PCR. *P<0.05 and **P<0.01 vs. liver cancer cell lines (Huh-7 and HepG2). IRF9, interferon regulatory factor 9; wks, weeks; p-, phosphorylated.