Fig 1. Doxorubicin modulates the expression levels of HBV, p21, and p53 in 1.3.ES2 cells.

(A) The effect of doxorubicin treatment on HBV replication. 1.3.ES2 cells were treated with different doses of doxorubicin for 1 hour, and then their culture medium was replaced with fresh culture medium. Total DNA, total RNA and cell lysates were obtained three days after doxorubicin treatment for 1 hour for Southern blot, Northern blot and particle blot/Western blot analyses, respectively. The expression levels of HBV transcripts, viral particles, HBcAg, p21, and p53 are shown. (B) The effect of doxorubicin on the modulation of viral particle generation. 1.3.ES2 cells were treated with 5 μg/ml doxorubicin for 1 hour then their culture medium was replaced with fresh culture medium for three days. Secreted viral particles were then obtained from the culture medium and analyzed to measure their content in terms of viral genomes by quantitative RT-PCR assay. (C) P21 participates in doxorubicin active HBV replication. 1.3.ES2 cells were infected with lentivirus as indicated for 24 hours, which was followed by doxorubicin treatment for 1 hour. The expression levels of total DNA, HBcAg, p21, and HBV transcripts were then analyzed by Western blot and Northern blot assays three days after doxorubicin treatment. Lane 1, control lentivirus expressing shLuc RNA. Lanes 2 and 3 are lentiviruses carrying shRNAs against p21 (sh-CDKN1A-A1 and sh-CDKN1A-G1, respectively). (D) The role of p53 in doxorubicin-mediated HBV activation. 1.3.ES2 cells were transfected with plasmid expressing a domain negative form of p53 (pCMV-p53DN) or mock vector (pCMV) and these two experimental groups were then subjected to doxorubicin treatment for 1 hour. The cell lysates were harvested three days after doxorubicin treatment. The expression levels of p21 protein and HBV transcripts were determined by Western blot and Northern blot analysis, respectively.