Fig. 4

CPS restores wtp53 activities in mutp53-carrying cells. a U373 and SKBR3 were treated with CPS (200 μM) for 24 h. Western immunoblotting was performed on equal amount of total cell extracts to detect phospho-Histone H2A.X (γH2AX) levels. Anti-β-actin was used as protein loading control. b SKBR3 and U373 cells (6x10⁶) were plated in 150 mm dish and the day after treated with CPS (200 μM) for 16 h before being assayed for chromatin immunoprecipitation analysis (ChIP) with anti-p53 or anti-p73 antibodies. PCR analyses were performed on the immunoprecipitated DNA samples using primers specific for wtp53 target gene promoter (Puma) or for mtp53 target promoter (MDR1). A sample representing linear amplification of the total chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunogloblulins (No Ab). SKBR3 and U373 cells were plated at subconfluence in 60 mm dish and the day after treated with CPS (200 μM) for 24 h, with or without autophagy inhibitor chloroquine (CHQ) (c) or p53 inhibitor pifithrin-α (PFT-α) (30 μM) (d). p53 target genes were detected by RT-PCR analysis. β-actin was used as control. Gene expression was measured by densitometry, normalized to β-actin levels, ±SD (right panels) and plotted as fold of mRNA expression over control (Mock)