Figure 4

Activation of a p53-dependent promoter and induction by Actinomycin D were normal in immortalized Bax-null myogenic cells. As indicated in panels A-F, wild-type, immortalized Bax-null, and immortalized p53-null myogenic cells were transfected with either PG13-GFP, a plasmid from which GFP expression requires normal p53, or with the control pCMV-β-Gal, a plasmid from which β-Galactosidase is expressed independently of p53. GFP was detected by endogenous green fluorescence, and β-Galactosidase was detected by immunocytochemistry with a specific antibody (red fluorescence). GFP was expressed in wild-type and Bax-null cells, but not in p53-null cells, whereas β-Galactosidase was expressed in all three cell types. Transfection efficiency, measured as percentage of β-Galactosidase-expressing cells, was independent of genotype and ranged from 5.0 – 15.7% in different experiments. Bar = 20 μm. For panel G, cultures of NIH3T3 cells, two immortalized Bax-null lines (BaxKO-1 and BaxKO-2), and one immortalized wild-type line (WT-1) were examined by immunoblotting for p53 expression. Expression of p53 was barely detectable in untreated cultures (ActD-), but was abundant in parallel Actinomycin D-treated cultures. Treated cells were exposed to 60 ng/ml of Actinomycin D for 8 hours and cells were collected for immunoblot analysis 16 hours after the Actinomycin D was removed and replaced with fresh medium.