Sc 81406

HepG2 cells with STAT3 or TP73 silencing and their control cells were cultured in 10 cm dishes. At 85% confluence, cells were fixed for 10 min using 10 mL of 1% formaldehyde solution freshly prepared with 1×PBS and subsequently quenched with 1 mL of 2.5 M glycine solution. After washing three times with 1×PBS solution, cell samples were harvested with cell scrappers and were sheared with an ultrasonication machine (Scientz 98-III,Ningbo, China). After sonicating for 20 min (pulse 5 s and interval 5 s) with 25% power, cell lysate was centrifuged for 10 min at 12,000 rpm, and the resulting supernatant was retained for ChIP assays as described previously (Zhang et al., 2022 (link)), where antibodies against TBP (SC-204; Santa Cruz Biotech.), BRF1 (SC-81405; Santa Cruz Biotech.), GTF3C2 (SC-81406; Santa Cruz Biotech.), and POL3RA (SC-292119, Santa Cruz Biotech.) were used in ChIP. The DNA from ChIP assays was purified with a QIAGEN PCR clean kit and eluted with 40 μL ddH₂O. 1 μL of ChIP DNA sample (1/40) was used for a qPCR reaction, while 0.02% input DNA (0.1 ng genomic DNA) was used in a qPCR reaction for positive control. Relative enrichment was obtained by comparing the quantity of target DNA in 1 μL of ChIP DNA sample to that in 0.02% input DNA.

The 293T cell lines stably expressing GATA4 shRNA or control shRNA were grown in 10 cm dishes. At 90% confluence, the cell lines were fixed for 10 min using a 10 mL PBS solution containing 1% formaldehyde and then quenched with 1 mL glycine solution (2.5 M). After washing three times with 1× PBS solution, the cells were suspended with 1 mL ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0) and disrupted with an ultrasonicator. After centrifugation at 12,000 rpm, the supernatant was retained and diluted 6 times with a ChIP dilution buffer (0.01% SDS, 1.1% TritonX-100, 1.2 mM EDTA, 16.7 mM Tris-HCl PH 8.0 and 167 mM NaCl). The rest procedures for ChIP assays followed, as described previously [43 (link)]. ChIP assays were performed using the antibody against BRF1 (SC-81405, Santa Cruz Biotech.), GTF3C2 (SC-81406, Santa Cruz Biotech.), and POLR3A (SC-292119, Santa Cruz Biotech.). After elution, the chromatin samples immunoprecipitated were subjected to de-crosslinking and DNA purification. The DNA from each ChIP sample was eluted with 40 μL ddH₂O. One μL of them (1/40) for individual factors was used for a qPCR reaction; meanwhile, 0.01% input DNA was used as a positive control. Relative occupancy was obtained by comparing the relative quantity of promoter DNA in 1 μL of ChIP sample to that in 0.01% input DNA.

HepG2, HuH-7, and 293T cells were cultured in 6-well plates. At 90% confluence, cells were harvested and lyzed with 1×SDS loading buffer. After heating at 100°C for 10 min, 20 μL cell lysate was loaded for SDS-PAGE electrophoresis and western blot analysis. Western blot was performed using antibodies against STAT3 (CST#9139, CST), mCherry (TAG0080, Frdbio), GAPDH (RAB0101, Frdbrio), BRF1 (SC-81405, Santa Cruz Biotech), GTF3C2 (SC-81406, Santa Cruz Biotech), GTF3C3 (SC-393235, Santa Cruz Biotech), TBP (SC-421, Santa Cruz Biotech), and TP73 (CSB-PA003696, CUSABio).