Figure 2.

Doxorubicin promotes RFX1 expression and HBV replication in parallel. (A) HepG2.2.15 and HepAD38 cells were treated with 1 μmol/L Dox for 1 h, respectively. The mRNA levels of RFX1 in each cell were quantified by qRT‐PCR at 48 h post‐Dox treatment. (B) The pBB4.5‐1.2 × HBV expression plasmid (2 μg) was transfected into HepG2 cells using lipofectamine 2000 in a 35‐mm dish. Then, the transfected HepG2 cells were treated with serial dosages of Dox for 1 h. The relative levels of 3.5 kb HBV RNA, RFX1, and CTBP mRNA in the cells were quantified by qRT‐PCR at 48 h post‐Dox treatment. ACTB mRNA was used as an internal control. The concentrations of Dox were 1: 0 μmol/L, 2: 0.05 μmol/L, 3: 0.1 μmol/L, 4: 0.2 μmol/L, 5: 0.5 μmol/L, 6: 1 μmol/L, and 7: 2 μmol/L. (C) The levels of RFX1 protein in HepG2 cells treated with or without 1 μmol/L Dox for 1 h were detected by Western blot at 48 h post‐Dox treatment (i). The right figure (ii) was the statistical graph of RFX1 protein levels detected by Western blot, which were analyzed by ImageJ software (NIH), at least in triplicate. The α‐tubulin was used as an internal control. (D) 1 × 10⁵ primary human hepatocytes (PHH) cells in a 48‐well plate were inoculated with 1 × 10⁷ copies of genome equivalent HBV in the presence of 4% PEG 8000 for 20 h. PHH cells were then washed with phosphate‐buffered solution (PBS) six times and maintained in PMM medium for 3 days. Then, the HBV‐infected PHH cells were treated with 1 μmol/L Dox for 1 h. The relative levels of 3.5 kb HBV RNA, total HBV RNA, and RFX1 mRNA in the cells were quantified by qRT‐PCR at 48 h post‐Dox treatment. ACTB mRNA was used as an internal control.