Fig. 1.

p53 negatively regulates FKHRL1 in response to genotoxic stress. (A) Increased phosphorylation of FKHRL1 after DNA damage in the presence of p53. Transformed WT and p53–/–MEFs were treated with UV radiation (30 J/m²), etoposide (0.5 μM), or TNF-α (10 ng/ml). Cell extracts were analyzed at the indicated time points by immunoblotting with antibodies directed against p53, p21, and phosphorylated FKHRL1. Actin was used as the loading control. For all figures, results shown are representative of at least three independent experiments. (B) p53-dependent and -independent apoptosis and G₂/M arrest caused by various stimuli. Transformed WT and p53–/–MEFs were treated for 24 h with the same DNA-damaging agents as in A, followed by propidium iodide (PI) staining and fluorescence-activated cell sorter analysis. Data are shown for the sub-G₁ (apoptosis) (Left) and G₂–M(Right) phases. Data are the means and variances for two independent experiments performed in duplicate. (C) Nuclear exclusion of FKHRL1 induced by p53 in response to DNA damage. p53–/–MEFs were cotransfected with an expression plasmid encoding HA-tagged WT FKHRL1 plus a plasmid encoding WT p53, p53R175H, or p53R273H. At 24 h posttransfection, cells were treated with UV radiation (30 J/m²) for 8 h. FKHRL1 was detected by immunofluorescent staining with anti-HA antibody. Nuclear DNA was stained with 4′,6-diamidino-2-phenylindole. The images were merged to detect nuclear localization/exclusion of FKHRL1. Results shown are one trial representative of three experiments. Quantification of the means and variances from three experiments is shown. (D) Decreased p27 expression in WT cells after DNA damage treatment. WT and p53–/–MEFs were exposed to UV radiation (30 J/m²) for 10 h or etoposide (0.5 μM) for 5 or 10 h. Cell lysates were analyzed by Western blotting with antibodies against p27 and actin. (E) Reduced association of FKHRL1 with the endogenous p27 and cyclin G2 promoters in the presence of p53 upon UV radiation. ChIP assays were performed on WT or p53–/–MEFs left untreated or treated with UV radiation (30 J/m²) for 10 h. PCR was conducted by using primers flanking murine p27 or cyclin G2 promoter regions containing FKHRL1-binding motifs. DNA templates from a protein–DNA complex immunoprecipitated with either FKHRL1-specific antibody (Upper) or no antibody (Lower, –Ab) were used. Input DNA was amplified for normalization (Lower).