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. 2017 May 2;11(6):612–627. doi: 10.1002/1878-0261.12051

Figure 2.

Figure 2

DIMP53‐1 shows a p53‐dependent growth inhibitory effect in human tumor cells mediated by cell cycle arrest, apoptosis, p53 stabilization, upregulation of p53 target genes, and disruption of the p53–MDM2/X interactions. (A) Concentration–response growth curves for DIMP53‐1 in p53+/+ and p53−/− HCT116, after 24‐ and 48‐h treatments; data are mean ± SEM of four independent experiments; incubation with DMSO, in equivalent % of DIMP53‐1, was used to normalize the results. (B,C) Cell cycle arrest (B) and apoptosis (C) were determined at 7 and 14 μm of DIMP53‐1 for 24 h in p53+/+ and p53−/− HCT116 cells; data are mean ± SEM of three independent experiments; values were significantly different from DMSO (*P < 0.05; **P < 0.01; ***P < 0.001). (D) mRNA levels of BAX and CDKN1A (p21) after 24‐h treatment with 7 and 14 μm of DIMP53‐1 in p53+/+ and p53−/− HCT116 cells; fold expression changes are relative to DMSO and correspond to mean ± SEM of three independent experiments. (E) Western blot analysis was performed after 24‐h (MDM2, p53) and 48‐h (PARP, BAX, PUMA, p21) treatments with 7 μm of DIMP53‐1 or DMSO in p53+/+ and p53−/− HCT116 cells. (F) p53 protein levels in HCT116p53+/+ cells treated for 24 h with DIMP53‐1 or solvent followed with cycloheximide (CHX; 150 μg/mL). (G) Co‐IP was performed with anti‐p53 (IP:p53) or anti‐immunoglobulin G (IgG) antibodies, followed by immunoblotting with anti‐MDM2, anti‐MDMX, and anti‐p53 antibodies in HCT116p53+/+ cells treated with 7 and 14 μm of DIMP53‐1 or DMSO for 8 h; whole‐cell lysate (input); in IP:p53 of MDM2, the cut top band corresponds to the anti‐p53 antibody, and the other two bands correspond to MDM2 isoforms. (H) Quantification of IP:p53 immunoblots; data are mean ± SEM of three independent experiments; values were significantly different from DMSO (*P < 0.05; **P < 0.01; ***P < 0.001). In (E), (F), and (G), immunoblots are representative of three independent experiments; GAPDH was used as loading control.