Phenotype-associated p53-responsive gene expression in IMR90 cells.

(A) Schematic of the p53-associated phenotypes. (B) Cell viability, senescence-associated ß-galactosidase activity (SA-ß-gal), and BrdU incorporation (mean ± SEM; n = 3) were measured for each condition as indicated in (A). In addition, DNA damage-induced senescence (DDIS) was included for comparison: cells were treated with etoposide (100 μM) for two days, and maintained for an additional five days in drug-free media. (C, D) Immunoblot analyses for the proteins indicated for total lysates and chromatin fractions from the cells as labeled. Cyclin A2, a cell cycle marker; HMGA proteins, senescence markers. d1 and d7 correspond to acDDR and DDIS, respectively (C). Core histones (C, D) and HMGA proteins (C) were stained with Coomassie blue. The arrow indicates non-specific bands (the Cyclin A2 blot in (C)). (E) Immunoblot analysis in the indicated cells for chromatin fractions for p53. sh and v, sh-p53#1 and corresponding lentiviral vector (a miR30 design), respectively. For acDDR, sh-p53 was introduced first for at least 5 days before administration of etoposide. For RIS and pApo, sh-p53 was introduced after the phenotype establishment. Core histones were stained with Coomassie blue. (F) Venn diagram showing the numbers of differentially expressed (DE) genes upon p53 depletion with lentivirus-mediated RNAi (sh-p53#1) compared to vector, in the indicated conditions. (G) Pathway heatmap for differentially expressed genes upon p53 depletion.