Figure 2

p53 family regulates expression of ISG20L1. (a) 293FT cells were transfected with vector control (pCEP4) or the indicated p53 family members for 24 h and then analyzed by qRT-PCR for ISG20L1. Results represent 3 independent experiments and error bars show standard error. (b) Experiment was performed as in (a) and Western analysis performed. Results are representative of 3 independent experiments. *The p63 antibody used cross-reacts with p73 due to sequence similarity in the region containing the epitope recognized by the antibody [65]. (c) p73 regulates ISG20L1, seen in Rh30 cells treated with paclitaxel (5 nM) and cisplatin (5 μg/mL) for times indicated. Right panel, Rh30 cells were infected with a pSico vector that expresses control virus (Con) or shRNA targeting all isoforms of p73. Results are representative of 3 independent experiments. (d) MDA-MB-231 cells were infected as above. Forty-eight h later, cells were treated with rapamycin (40 nM) and RNA harvested for real-time analyses 24 h after treatment. Results represent 3 independent experiments and error bars show standard error. (e) Antibodies specific to p53, p53-Ser15 (S15), p63, and Bax (negative control) were used for ChIP analysis of the ISG20L1 promoter under control and cisplatin-treated (24 h, 10 μg/mL) conditions in HMECs. The p53 binding site in the p21 promoter and a region containing no p53 binding sites in the GAPDH promoter serve as positive and negative controls, respectively. Experiment is representative of duplicate independent templates. (f) ChIP analysis of p73 binding to the ISG20L1 promoter in Rh30 cells treated with rapamycin (40 nM) for 24 h.