Figure 3.
Cohesin inhibits HBV replication. (A) Huh7 cells were transfected with Flag-SMC3 or siSMC3 for 72 hours, the expression of cohesin complex subunits was measured with Western blot. (B) Huh7 cells were transfected with MC-HBV for 3 days, while PHH, HepaRGNTCP, and HLCZ01 cells were infected with HBV at 200 genome equivalents (Geq) for 7 days. Then, cccDNA was extracted using a Hirt protein-free DNA extraction procedure and subjected to Southern blot assay. The cccDNA from the HepAD38 cell line was taken as a positive control. (C and H) In the MC-HBV/Huh7 model, MC-HBV was cotransfected with Flag-SMC3 or siSMC3 into Huh7 cells for 72 hours; in HBV-infected HLCZ01, PHH, and HepaRGNTCP models, cells were infected with HBV at 200 Geq for 24 hours and then transfected with Flag-SMC3 or siSMC3 for another 5 days, HBV replication was analyzed through measuring HBsAg/HBeAg (enzyme-linked immunosorbent assay), pgRNA, HBV DNA, and HBc antigen (HBcAg) (Western blot) (n = 3). (I) Huh7 cells were transfected with Flag-SMC3 and MC-HBV for 72 hours, and immunofluorescence staining was performed to detect Flag-SMC3 and HBsAg expression levels. Scale bar: 20 μm. (J) Huh7 cells were transfected with MC-HBV and siSMC1A or siRAD21 for 72 hours, and viral replication was analyzed (n = 3). Data are presented as means ± SD. ∗P < .05, ∗∗P < .01. DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PF-DNA, protein-free DNA.