Figure 3

Up-regulation of the hTERT gene transcription in cells overexpressing HBZ and JunD. HeLa cells were cotransfected with expression plasmids for HBZ and JunD and incubated for 48 hours. (A) Western blot analysis of cell lysates using appropriate antibodies; lane 1, mock transfected; lane 2, JunD-transfected; lane 3, HBZ-transfected; lane 4, HBZ/JunD-transfected. (B) RT-PCR analysis of mRNA extracted from transfected cells. The RNAs were isolated and reverse transcribed. PCR was performed using primers specific for hTERT and actin. (C) Real-time PCR quantification of hTERT mRNA expression from cells transfected with indicated plasmids was performed as described in "Materials and methods". The expression level in mock-transfected cells was defined as 1.0. Experimental variations are indicated by error bars. (D) Recruitment of HBZ and JunD to the hTERT proximal promoter by chromatin immunoprecipitation assay (ChIP) in HeLa cells overexpressing HBZ and JunD. PCR results from IP reactions using preimmune rabbit serum (IgG) and antibodies against HBZ and JunD are shown. Each panel shows amplification of 0.4% of the total input chromatin (input). Purified DNA was analyzed by PCR using primers spanning the hTERT proximal promoter (upper panel) or the hTERT distal promoter (lower panel). DNA size standards are indicated. Data are shown for one representative experiment from three independent assays.