Fig. 4

Down-regulation of DAXX represses HPV genome transient replication. U2OS cells were first transfected with Daxx siRNA or a control siRNA, and 24 h later with wt HPV18 (a) or wt HPV11 (b) genome. Cells were harvested 72 h after transfection, episomal DNA was isolated, digested with DpnI and linearizing enzyme, EcoRI or BamHI, and analyzed by Southern blotting using a radiolabeled probe specific for the viral genome. The linearizing enzyme (M1) and DpnI (M2) digested pBRHPV18 or pUCHPV11 plasmids as markers are shown on the right. c qPCR analysis of the episomal DNA isolated in replication assays. Episomal DNA was digested with DpnI and analysed by qPCR using HPV18 or HPV11 genome, and mitochondrial DNA specific primers. The data represent the average of three independent experiments and are presented graphically relative to the basal replication level of the HPV18 or HPV11 genome. d Western blot analysis of the DAXX protein levels in the cells used in replication assays. Cells (10⁵) were lysed, separated electrophoretically, and immunoblotted with anti-DAXX and anti-tubulin antibodies