FIG. 4.

p53 and its target genes are activated in hAFS cells after DNA damage. (A) hAFS cells were cultured in chamber slides and treated with 50 μM etoposide for 20 h (Eto treated), or left untreated for control. The cells were fixed and stained with an antibody directed against γ-H2AX (red). The cell nucleus was stained with DAPI (blue). (B) Undifferentiated hAFS cells were treated with 50 μM etoposide for 19 h (UD + Eto), or with DMSO for control (UD + DMSO). Some of the hAFS cells were differentiated for 14 days and then treated with 50 μM etoposide for 20 h (D + Eto), or with DMSO for control (D + DMSO). The cells were harvested and subjected to western blotting to monitor the abundance of p53 and Mdm2. β-actin was used for loading control. (C) hAFS cells were transfected in duplicates with a siRNA targeted against p53 (p53sir) or with a control siRNA (Csir). Forty-eight hours after transfection, cells were treated with etoposide (50 μM f.c.) for further 20 h. Cells were harvested and one of the duplicates was used to monitor p53 abundance by western blotting. GAPDH was used for loading control. From the second duplicate, RNA was purified and transcribed into cDNA. The cDNA was analyzed by qRT-PCR for the abundance of p21 and mdm2. Abundance of specific cDNAs was normalized by determining the abundance of the housekeeping gene β-actin. Relative abundance of the specific RNA in the cells with control siRNA was set to one. The graph shows mean values and standard deviations of three independent experiments. (D) hAFS cells were transfected with a siRNA targeted against p53 or with a control siRNA for 2 days. Thereafter, some of the cells were treated with etoposide (50 μM f.c.) for 20 h. Cells were lysed and analyzed for PARP, for cleaved PARP, and for p53 abundance. β-actin was used for loading control. PARP, poly ADP-ribose polymerase. Color images available online at www.liebertpub.com/scd