Journal: Cell Communication and Signaling : CCS

Article Title: ZNF638 represses the transcription of HBV closed circular DNA involving HUSH complex-mediated histone modifications of epigenetic silencing

doi: 10.1186/s12964-026-02726-1

Figure Lengend Snippet: ZNF638 knockdown modulated histone modifications of HBV cccDNA microchromosomes. A Nuclear HBV cccDNA pulldown-WB procedures. After transfection with siRNAs targeting ZNF638, TASOR, and SETDB1 for 48 h, HepAD38 cells were transferred to Tet-free medium to induce HBV replication and cultured for an additional 6 days; HepG2-NTCP cells were infected with 6 × 10 5 copies/ml HBV for 6 days. T5 exonuclease was used to digest isolated extracts to remove rcDNA, ssDNA and dsDNA. Following incubation with a specific biotinylated cccDNA bait, streptavidin-coated magnetic beads were employed to precipitate the baited cccDNA and associated proteins in the complex. B Western blotting for the detection of proteins of interest alone with various histone H3 in HepG2-NTCP cells (left) and HepAD38 cells (right). Semiquantitative measures from densitometry were normalized to controls and plotted as changes in folds (n = 3). C Southern blotting for HBV cccDNA from HepG2-NTCP (left) and HepAD38 (right) cells. (Nondig.: Non-digested DNAs)

Article Snippet: The probing antibodies against the following antigens: ZNF638 (Bethyl, USA), MPP8 (Proteintech), H3K9me3 (Abcam, UK), H3K27ac (Gene Tex, San Antonio, USA), H3K4me3 (Active motif, Carlsbad, CA, USA), H3 (Cell Signaling Technology, Massachusetts USA), SETDB1 (Proteintech) and GAPDH (Proteintech) were used for overnight incubation at 4 °C following the blocking procedure.

Techniques: Knockdown, Transfection, Cell Culture, Infection, Isolation, Incubation, Magnetic Beads, Western Blot, Southern Blot

Journal: Cell Communication and Signaling : CCS

Article Title: ZNF638 represses the transcription of HBV closed circular DNA involving HUSH complex-mediated histone modifications of epigenetic silencing

doi: 10.1186/s12964-026-02726-1

Figure Lengend Snippet: ZNF638 dependent epigenetic silencing of HBV cccDNA transcription required marking of SETDB1-mediated H3K9me3. A Levels of HBV DNA, cccDNA and pgRNA in SETDB1 knockdown HepG2-NTCP cells at 6 d of 6 × 10 5 copies/ml HBV infections after SETDB1 siRNA transfection for 48 h. B Dose response of SETDB1 knockdown on increased HBV DNA levels. C Time course of HBV cccDNA transcription shown as the ratio of pgRNA to cccDNA for efficacy comparison. D-G Effect of SETDB1 siRNA transfection at 48 h on normalized HBV transcription levels at 6 d of 6 × 10⁵ copies/ml HBV infections in ZNF638 knockdown or overexpressing HepG2-NTCP cells. H-I ChIP-qPCR assays on cccDNAs for H3K9me3 enrichment at the ZNF638 binding regions using cccDNAs isolated from sgZNF638 or ZNF638oe HepG2-NTCP cells at 6 d post HBV infection

Article Snippet: The probing antibodies against the following antigens: ZNF638 (Bethyl, USA), MPP8 (Proteintech), H3K9me3 (Abcam, UK), H3K27ac (Gene Tex, San Antonio, USA), H3K4me3 (Active motif, Carlsbad, CA, USA), H3 (Cell Signaling Technology, Massachusetts USA), SETDB1 (Proteintech) and GAPDH (Proteintech) were used for overnight incubation at 4 °C following the blocking procedure.

Techniques: Knockdown, Transfection, Comparison, ChIP-qPCR, Binding Assay, Isolation, Infection