Fig. 1.
p53 is activated by Set7/9 by a methylation-independent mechanism. (A) Expression plasmids for pG13L, p53WT, p53K372R, and Set7/9 were cotransfected into HCT116 (p53−/−) cells as indicated. Cells were harvested 24 h after transfection, and relative luciferase activity was measured. The luciferase activity was normalized to the amount of protein in the cell lysate. The cell sample transfected with p53WT alone served as the control and its value (relative luciferase activity) was set as 1. The relative luciferase activities of the other samples are normalized to this control. Data are means ± SD (n = 3). (B) An empty plasmid and an expression plasmid for p53WT or p53K372R were cotransfected with or without Set7/9 into HCT116 (p53−/−) cells and, after 24 h, cells were treated with 1 μM Adr for 6 h, and a quantitative PCR was performed to analyze p21Waf1/Cip1 expression. The signals were normalized to the expression of GAPDH. (C) An expression plasmid for p53WT or p53K372R was cotransfected with or without Set7/9 into HCT116 (p53−/−) cells treated under the same conditions as in B. Cells were harvested and subjected to a q-ChIP assay with anti-p53 to measure the DNA binding ability of p53 on the p21Waf1/Cip1 promoter. (D) Expression plasmids for p53K372R, HA-p300, Myc-SIRT1, and Flag-Set7/9 were cotransfected into HCT116 (p53−/−) cells and, after 24 h, derived cell lysates were subjected to Western blotting by using anti–acetyl-p53 (K382), anti-p53, anti-HA, anti-Set7/9, or anti-SIRT1. GAPDH served as the loading control. (E) HCT116 (p53+/+) cells were transfected with a nonspecific siRNA (lanes 1 and 5), an siRNA against Set7/9 (lanes 2 and 6), an siRNA against SIRT1 (lanes 3 and 7), or siRNAs against both Set7/9 and SIRT1 (lanes 4 and 8). Twenty-four hours after transfection, cells were treated with 1 μM Adr for 6 h, and the same lysates were subjected to Western blotting by using anti–acetyl-p53 (K382), anti-p53, anti-Set7/9, or anti-SIRT1. GAPDH served as the loading control.