Fig. 3

Effects of DNA-damaging drugs and inhibitors of transcription on the viability of normal human fibroblasts and expression of p53 and its effectors

Human primary fibroblasts (GM03348C) were treated with medium only (untreated, untr.) or with medium containing cisplatin (CisPt, 10 µM), actinomycin D (ActD, 0.4 µM), α-amanitin (αAM, 1 µM), and DRB (DRB, 100 µM) for 12 hr (marked by an arrow on the x-axis of the cell viability graph). The cells were then washed, refed with fresh medium, and incubated for 84 hr more. Cell samples for protein analysis (A, Western blots on the left side of the figure), MTT-based cell viability assays (A, graph on the right side of the figure) and FACS scan analysis (B) were harvested at the indicated time points. Equal amounts of total protein were loaded onto SDS Polyacrylamide gels and Western blots were probed with antibodies that recognize p53, p21^WAF1,Cip1, and Mdm2 (only the corresponding parts of the Western blots are shown). Arrowheads on the right side of Mdm2 Western blots indicate Mdm2. The values in the cell viability graphs are averages from two independent experiments with a difference of less than 20%. (B) Cell cycle analysis of untreated (untr.) and actinomycin D (ActD)-treated cells after 96 hr. DNA content is shown on a logarithmic scale. The sub-G1 area (D) contains apoptotic cells.