Fig 2: The impacts of HBc on IFNα anti-virus activity in vitro. (a) HepG2 cells were transfected with BAF200 with or without HBc and stimulated with 500 U/mL IFNα for indicated time. (b) Deletion efficiency of siRNA-oligos were examined in HepG2 cells using western blot. pGC-FU-Flag vectors, siRNA, or pGC-FU-Flag-BAF200 pasmids were transfected into HepG2 cells (c) or HepG2.2.15 cells (d,e), then treated with IFNα in indicated concentration for 24 h. Afterwards, the cell viability was measured using MTT assay and direct cell counting via trypan blue exclusion assay (a,c–e). (f) HepG2 cells were transfected with pHBV1.31 and pGC-FU-Flag-BAF200, or co-transfected with pCMV-HA-HBc, then treated with 500 U/mL IFNα for 72 h. HBV replication levels were determined in the supernatants by measuring HBsAg and HBeAg concentration using ELISA and HBV DNA copy numbers using RT-qPCR. The empty vector and pHBV1.31 were co-transfected in the non-IFNα induced condition as control. The control without IFNα treatment was designed as 100% (a,c–f). *, p < 0.05, **, p < 0.01, NS = non-significant.

Fig 3: HBc regulates IFITM1 expression by binding to BAF200. (a) HepG2 cells were transfected with pGC-FU-Flag vector or pGC-FU-Flag-BAF200, treated with IFNα (500 U/mL) or not for 18 h, then IFITM 1, 2, and 3 expression levels were measured via western blot using antibodies against IFITM 1, 2, and 3 and normalized to the GAPDH mRNA level. Results are standardized to a value of 1 for IFITM2 mRNA level of empty vector transfection control without IFNα treatment. (b) Total RNA was extracted and the mRNA levels of IFITM 1, 2, and 3 were detected via RT-qPCR. (c) HepG2 cells were transfected with control siRNA (siControl) or siBAF200 and then treated with IFNα (500 U/mL) for 18 h. Levels of IFITM1 and β-actin proteins were determined by western blot using the corresponding antibodies. (d) The effect of HBc overexpression on IFITM1 expression levels was measured by western blot assays. (e) IFITM1 transcription levels under induction of IFNα at different concentrations were examined via RT-qPCR assays, and normalized to the GAPDH mRNA level. The vector transfection control treated with 1000 U/mL IFNα was designated as 100%. *, p < 0.05; **, p < 0.01.

Fig 4: SMARCA4 mutational spectrum in the FoundationCORE® patient cohort.a SMARCA4 alteration frequency separated by disease ontology (n = 131,668 patients). b Distribution of SMARCA4 mutation types (n = 10,562 variants). c Zygosity of SMARCA4 truncating and nontruncating variants in patients where zygosity was determined (n = 5699 patients). d Mutual exclusivity of SMARCA4 mutations with the aggregate of other BAF genes profiled in the FoundationOne® panel (ARID1A/B, ARID2, PBRM1, SMARCB1, SMARCD1).

Fig 5: HBc downregulates IFITM1 expression by modulating PBAF stability. (a) The pCMV-Flag vector and pCMV-Flag-BAF200 were transfected into HepG2 cells, with or without IFNα (500 U/mL) treatment for 24 h. Phosphorylation of STAT1 was detected via western blot using antibodies against phosphorylated STAT1 (pSTAT1). Meanwhile, STAT1 and BAF200 were detected using anti-Flag and anti-STAT1 antibodies as control. (b) HepG2 cells were co-transfected with pGC-FU-Flag-BAF200 and pCMV-HA vector or pCMV-HA-HBc, with or without IFNα (500 U/mL) treatment for 24 h. Co-IP assays were performed with anti-Flag antibody, then BAF200–BAF180 immuno-complexes were detected by western blot assays using anti-BAF180 antibody or anti-Flag antibodies (control). Input control assays were performed in WCL using anti-HA or anti-BAF180 antibodies. Meanwhile, total mRNA was extracted to examine the effect of BAF200 and HBc combination on IFITM1, 2, 3 transcriptions by RT-PCR (c). β-actin was detected as control. IFITM1 transcription level was determined using RT-qPCR and normalized to the GAPDH mRNA level. (d) The Flag-BAF200 transfection with IFNα treatment was designated as 1. *, p < 0.05; **, p < 0.01. ND = Not Detected. (e) HepG2-NTCP cells were incubated with supernatants isolated from the supernatants of HepaAD38 cells cultures (containing HBV) at 1000 GEq for 16 h or not (mock). Co-IP assays were performed with BAF200 antibodies, and immuno-complexes were detected by western blot assays using BAF180 antibodies or BAF200 antibodies. Input control assays were performed in WCL using HBc antibody or BAF180 antibody.