Fig 2: The CTCF-mediated cohesin recruitment on cccDNA is necessary for cohesin-initiated inhibition on HBV replication. (A) MC-HBV was cotransfected with Flag-SMC3 and siCTCF into Huh7 cells for 72 hours, and HBsAg, HBeAg, HBc, pgRNA, and HBV-DNA levels were measured (n = 3). (B) The localization of CTCF-BS in the HBV genome and their sequence of WT and synonymous mutants. ※The CTCF-BS consensus sequence. (C) MC-HBV and CTCF-BS mutants were transfected into Huh7 for 72 hours, and the enrichment of CTCF on cccDNA was analyzed by ChIP (n = 3). (D) Sequence alignment of CTCF-BS1 and BS3 with different genotypes of HBV strains using the Clustal W algorithm of DNAstar Megalign software (Madison, WI). The dot represents the identical nucleotide; the red box indicates CTCF-BS1 and BS3 sequences. (E) MC-HBV and CTCF-BS mutants were transfected into Huh7 for 72 hours. The cccDNA binding of SMC3 was analyzed by ChIP (n = 3). (F) Flag-SMC3 was cotransfected with WT MC-HBV and CTCF-BS mutants (mBS1, mBS3) into Huh7 cells for 72 hours, HBsAg/HBeAg expression and pgRNA transcription were measured (n = 3). (G) WT, CTCF-mBS1, and CTCF-mBS3 mutants of MC-HBV were transfected into Huh7 for 72 hours, HBsAg/HBeAg expression and pgRNA transcription were measured (n = 3). Data are presented as means ± SD. ∗P < .05, ∗∗P < .01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig 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.

Fig 4: The relationship between METi and DNA damage‐related phosphorylations in vivo. IHC assessment in a xenograft tumor model of EBC‐1 cells. Animals were treated by oral administration of METi (15 mg·kg−1·day−1 for six consecutive days), local irradiation [a single dose (6 Gy) on day 3], or the combination of these two modalities. Following these treatments, the levels of mentioned proteins or phosphorylations sites were assessed in tumor tissues. Representative images (left panel) and quantification [% of positively stained area; mean (n = 3) ± SEM; right panel] of (A) MET Y1234&5, NUMA1 S395, (B) ATM S1981, SMC3 S1083, and (C) CHEK1 and CHEK1 S345 in EBC‐1 subcutaneous xenografts following IR, METi, or their combination. Statistical analysis was performed with the GraphPad Prism Software using one‐way ANOVA test (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig 5: KDAC6 and KDAC8 activity persists despite treatment with selective KDAC inhibitors. Cell lines were treated with either Tubastatin A (Tuba) or PCI‐34051 (PCI) for 2 days to compare acetylation levels of previously characterized substrates. (A) Representative immunoblots of lysates showing levels of SMC3 K105/K106 acetylation for each condition. Total SMC3 signal served as a loading control. Top and middle panels are different exposure levels of the same blot to allow for comparison of both weak and strong bands. (B) Representative immunoblots of lysates showing levels of α‐tubulin K40 acetylation for each condition. Total α‐tubulin signal served as a loading control. Top and middle panels are different exposure levels of the same blot to allow for comparison of both weak and strong bands. (C) Representative images of immunofluorescence of fixed cells using antibodies against α‐tubulin (magenta in merge) and α‐tubulin acetylated at K40 (green in merge). Gray images of each channel are normalized to the same maximum intensity. The intensity range of each channel was individually maximized for merged images, to more effectively show relative localization (white). Scale bars represent 20 μm. (D) Signals from immunofluorescence images were quantified for both α‐tubulin and acetyl‐K40 α‐tubulin. The ratio of acetyl‐tubulin/tubulin was calculated for individual cells (n ≥ 45 for each group). Whiskers represent the mean and standard deviation. Significance was evaluated by multiple t‐tests with Bonferroni correction.