Effect of site-specific mutagenesis and DNA damage on p53-mediated transactivation of FATS. (A) Site-directed mutagenesis in p53-RE consensus to generate M-746-luc. (B) The reporters was tranfected alone, or co-transfected with increased amount of p53 (0.1 μg, 0.3 μg, respectively) into U87 cells. Dual luciferase activities were measured after 24 h. (C) Luciferase reporter assay in p53-null cells. (D) MEF cells were untreated or treated with etoposide (0.1 μg/ml) for 4 h. The total RNA was prepared and the expression of FATS mRNA was subsequently detected by RT-PCR. A negative control (Ctr.) with no reverse transcriptase was also showed. (E) MEF cells were untreated or treated with etoposide (0.1 μg/ml) for 6 h. Cell lysates were prepared and subjected to immunoblotting. (F) U87 cells were untreated or treated with ionizing radiation (IR, 10Gy), UV (4 kJ/m2), or actinomycin D (Act.D, 20 nM), respectively. The expression of FATS mRNA was detected by RT-PCR. (G) Induction of FATS protein in p53-null MEF cells with or without etoposide (0.1 μg/ml) treatment for 4 h.