Up-regulation of DR4 and DR5 expression by ANT2 shRNA-induced activation of p53 in MCF7 cells. (A) Immunoblot analysis of p53 expression and phosphorylation. MCF7 cells were transfected with scrambled shRNA or ANT2 shRNA. After 24 h, cells extracts were prepared, subjected to SDS-PAGE, and immunoblotted with antibodies against phospho-p53, p53, α-tubulin, ANT2, and β-actin (control). Band intensities were calculated using Image Gauge v4.0 (Fujifilm) (B) Reporter gene analysis of p53 transcriptional activity. MCF7 cells were transfected with a p53-luciferase-reporter construct and scrambled or ANT2 shRNA. After 18 h, cell lysates were prepared and analyzed for luciferase activities using a luminometer. (C) Immunoblot analysis of p53-mediated up-regulation of DR4/DR5. Cells were pre-treated with the p53 inhibitor pifithrin-α for 2 h and then transfected with scrambled or ANT2 shRNA. After 24 h, cell lysates were prepared, subjected to SDS-PAGE, and immunoblotted with antibodies against DR4, DR5, and α-tubulin. (D) Cells were treated with pifithrin-α and scrambled or ANT2 shRNA as in (C) and then analyzed for cell viability using a CCK8 assay kit.