Figure 7.

Chk1 and Chk2 regulate TAp73 promoter activation by E2F1. (A, upper panel) H1299 cells were transfected in the presence or absence of 150 nM UCN-01 with constructs expressing Myc-E2F1 (100 ng), HA-DP1 (100 ng), p73-Luc (500 ng), and a pCMV-RL construct (10 ng) for 24 h. (Lower panels) Luciferase assays were performed by standard methods and data were normalized to control Renilla luciferase signal. A representative immunoblot of MycE2F1, HA-DP1, and actin. (B, upper panel) shE2F1 H1299 cells were transfected with the indicated siRNA as in Figure 1, then transfected with 5 ng HA-E2F1, 1 μg p73-Luc, and 10 ng pCMV-RL constructs for 24 h. Luciferase assays were performed by standard methods and data were normalized to control Renilla luciferase signal. (Lower panels) A representative immunoblot of HA-E2F1, Chk2, and actin. (C, left panels) H1299 cells were transfected as in Figure 1, and treated with 10 μM VP16 for 24 h. Chromatin immunoprecipitation was performed using an anti-E2F1 antibody (C20). Precipitated DNA was amplified with primer pairs for each promoter as indicated for p73 and Thymidine Kinase (TK). Nonspecific amplification was controlled for by performing PCR of a region within the GAPDH promoter. (Middle panels) Input DNA PCR products (1% of total). (Right panels) Immunoblotting of cross-linked protein extracts for E2F1 and Actin. (D, left panels) H1299 cells were exposed to 150 nM UCN-01 for 1 h prior to the addition of 10 μM VP16 or 300 nM CPT for 24 h. Chromatin immunoprecipitation was performed as in C. (Right panels). Input DNA samples as in C.