Fig. 2.

EMS is a direct target gene of p53. (A) Schematic illustration of four putative repressive p53 response elements (RE1, RE2, RE3, and RE4) in the EMS gene promoter. (B) A549 cells were cotransfected with control vector, Flag–p53, or together with the reporter constructs in the indicated combination. Twenty-four hours after transfection, reporter activity was measured. Data shown are mean ± SD (n = 3). **P < 0.01. (C) A549 cells expressing either control shRNA or p53 shRNA were transfected with the indicated reporter constructs. Twenty-four hours after transfection, reporter activity was measured. Data shown are mean ± SD (n = 3). *P < 0.05. (D and E) A549 cells expressing either control shRNA or p53 shRNA were cotransfected with pGL3-EMS-P and Renilla luciferase plasmid. Twenty-four hours after transfection, cells were treated with (D) 10 μM Nutlin or (E) 10 μM Etoposide for 24 h. Reporter activity was then measured. Data shown are mean ± SD (n = 3). **P < 0.01. (F) A549 cells were cotransfected with control vector, Flag–p53, or together with the indicated reporter constructs. Twenty-four hours after transfection, reporter activity was measured. Data shown are mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. (G) Lysates from A549 cells were subjected to ChIP assay using anti-p53 antibody or an isotope-matched control IgG. ChIP products were amplified by PCR. ctrl, control; IgG, immunoglobulin G; IP, immunoprecipitation; ns., no significance.