Fig 2: The level of total viral DNAs, including 2-LTR circles, increases over time in the absence of CHAF1A. (A and B) Time-course analyses were performed with CHAF1A KD or NT-ctrl cells infected with integration-deficient (IN-D64A) reporter virus and qPCR analyses were used to determine levels of total viral DNA (A) or 2-LTR circles (B). Data are shown relative to GAPDH levels. (C–F) CHAF1A KD or NT-ctrl cells were infected with integration-deficient (HIV-1_IN-D64A) or integration-proficient (IN-wt) reporter viruses as indicated and DNA was isolated 24 h after infection. qPCR analyses were performed with ZsGreen-specific primers to measure total viral DNA (C and D) or with primers specific to the LTR junction region of 2-LTR circles (E and F). Data are depicted relative to GAPDH levels. ns, not significant, P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. Data are expressed as mean ± SD; n = 3 to 4 independent experiments. Mock represents uninfected control.

Fig 3: The effect of growth arrest after CHAF1A knockdown in HepG2 and Hep3B cells.Notes: (A) HepG2 and Hep3B cells that had been transfected with CHAF1A shRNA or nontarget control shRNA (control), respectively, were subjected to qRT-PCR. **P<0.01 by paired sample t-test; n=3 repeats with similar results. (B) HepG2 and Hep3B cells that had been transfected with CHAF1A shRNA or nontarget control shRNA (control), respectively, were subjected to Western blotting. (C) Cell proliferation as assessed by BrdU cell proliferation assays was inhibited by CHAF1A shRNA in HepG2 and Hep3B cells. **P<0.01 by t-test; n=3 repeats with similar results. (D) As assessed by MTT assays, the CHAF1A knockdown group was found to reduce the viability of HepG2 and Hep3B cells. **P<0.01 by two-way ANOVA; n=3 repeats with similar results. (E) Representative results of colony formation of HepG2 and Hep3B after transfection. **P<0.01 by t-test; n=3 repeats with similar results. (F) Expression of Ki-67 was verified by immunofluorescence staining after transfection. Merged pictures are overlays of both Ki-67 green signals and nuclear staining by DAPI (blue). Magnification ×400. (G) Cell cycles determined in HepG2 and Hep3B cells after transfection. Histogram indicates the percentage of cells at G0–G1, S, and G2–M cell cycle phases. **P<0.01 by t-test; n=3 repeats with similar results. Values are depicted as mean ± SEM.Abbreviations: ANOVA, analysis of variance; BrdU, 5-bromodeoxyuridine; CHAF1A, chromatin assembly factor 1, subunit A (P150); DAPI, 4′,6-diamidino-2-phenylindole; qRT-PCR, quantitative real-time polymerase chain reaction; MTT, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; SEM, standard error of the mean; OD, optical density; RLU, relative light unit.

Fig 4: Depletion of CHAF1A enhances NEIL1 initiated SN-BER but not LP-BER. (A) Western analysis of CHAF1A and CHAF1B in doxycycline (Dox) inducible CHAF1A shRNA expressing U2OS cells. (B) Schematic representation of the substrate oligonucleotide indicating incorporation of radiolabelled [α-32P]dCMP reflecting SN-BER, while incorporation of [α-32P]dTMP indicates LP-BER resulting from strand displacement 3′ to the lesion site (5-OHU) with replicated patch of four nucleotides (10,20). (C) Effect of CHAF1A on NEIL1 initiated SN-BER. NE from control and CHAF1A downregulated cells with or without GOx treatment were isolated for the assay, as described in Materials and Methods. (D) Effect of CHAF1A on NEIL1 initiated LP-BER as in C. (E) SN-BER activity in FLAG-NEIL1 IP complex isolated from control and CHAF1A downregulated cells. Other experimental details are described in Materials and Methods. (F) LP-BER assay with FLAG-NEIL1 IP complex. Relative quantification of repaired product are shown (with P values; Student's t Test). (G) Western analysis of FLAG (NEIL1), CHAF1A and CHAF1B levels in the FLAG IPs of cell lysates from control and CHAF1A downregulated cells.

Fig 5: CHAF1A participates in the regulation of c-MYC and CCND1 in GC. a–c Correlation of CCND1, c-MYC, CDK6 with CHAF1A mRNA expression in 45 pairs of GC and adjacent non-tumor tissues. The data were derived from the GEO database (GSE63089). d RT–PCR analysis of CCND1, c-MYC, CDK6, CCNE1, CDK2 and CDK4 mRNA expression in BGC-823 cells transfected with CHAF1A or control plasmid. e Western blot analysis of c-MYC and CCND1 level in BGC-823 cells that transfected with CHAF1A or control plasmid. f–g The mRNA expression of c-MYC and CCND1 in HGC-27 and MGC-803 cells transfected with CHAF1A siRNA or negative control. h–i The protein expression of c-MYC and CCND1 in HGC-27 and MGC-803 cells under CHAF1A knockdown. j–k Western blot analysis of the global H3K9me3 levels in HGC-27 and MGC-803 cells transfected with CHAF1A siRNA or negative control. l Western blot analysis of the global H3K9me3 levels in BGC-823 cells transfected with CHAF1A or control plasmid.