Overexpressed p53 neither causes cell cycle arrest nor inhibits growth even though it exhibits in vitro as well as in vivo DNA binding. (A) Cells treated with 100 and 1000 ng/ml of Dox were incubated for 48 h and processed for flow cytometric analysis by PI staining. Graphical representation of FACS data, bar graph represents % cell populations in each cell cycle phase (± S.E.). (B) Five hundred cells seeded in 35 mm plates and treated with Dox as mentioned in (A) were allowed to grow for 21 days with replacing Dox containing medium every 4 day. Cells in plates were stained with crystal violet stain and colonies were counted. Bar graph represents average of colony number per plate (± S.E.) from three independent experiments. (C) For in vitro DNA binding, after 100 and 1000 ng/ml Dox treatment for 48 h, nuclear extracts derived from HTet23p53, HTet26p53, HTet43GFP and HeLa cells were incubated with a γ-32P-labeled DNA probe having the consensus p53 binding motif. Bar graph represents densitometric values of autoradiograph (± S.E.). (D) In vivo transactivity of p53 was determined by transfecting pG13CAT as well as pEGFPC1 plasmid in HTet23p53, HTet26p53, HTet43GFP and HeLa cells following Dox treatment of 48 h. Percentage CAT activity was calculated by measuring the acetylations of 14C-chloramphenicol on thin layer chromatography using phosphorimager. Percent CAT/GFP for HTet43GFP was calculated by dividing with GFP reading for without Dox treated cells only. PFTα was used for the specificity of the p53 activity. Bar graph represents %CAT values normalized to GFP fluorescence for transfection efficiencies from three independent experiments (± S.E.).