Fig 2. Expression level of p21 modulates HBV replication in 1.3.ES2 cells.

(A) Cell cycle analysis of 1.3.ES2 cells with p21 overexpression. 1.3.ES2 cells were transduced with AdIE (a mock control adenovirus) or with AdIE/p21 (an adenovirus carrying the p21 gene). Next the cell cycle distribution was analyzed by flow cytometry assay. (B) The effect of p21 overexpression on the modulation of HBV replication. Cell lysates were extracted from adenoviruses-infected 1.3.ES2 cells, and the levels of HBV genomes and HBV transcripts were examined by Southern and Northern blot analysis using a HBV-specific probe. The expression of beta-actin was used as the loading control in the Northern blot. The expression of HBV nucleocapsids and the embedded viral genome were detected by particle blot analysis. The expression levels of HBcAg and p21 were analyzed by Western blot assay. (C) The dosage effect of p21 on the modulation of HBV replication. 1.3.ES2 cells were transduced with different amounts of AdIE and/or AdIE/p21, and equal amounts of cell lysates were subjected to native agarose gel electrophoresis to allow particle blot analysis. The encapsidated viral genomes were detected using a HBV-specific probe. (D) Effect of p21 knock-down on the modulation of HBV replication. 1.3ES2 cells were infected with lentiviruses that expressed control shRNA or shRNA against p21. Four days after lentivirus infection, the cells were harvested for Northern blot and Western blot analysis. Lanes 1, 2 and 3 contained control virus samples (vector alone, shRFP and shLuc, respectively), while Lanes 4 and 5 contained cells transfected with two different shCDKN1As; these were sh-CDKN1A-A1 and sh-CDKN1A-G1, respectively.