Fig. 4: Acetylation of mutp53 at the DBD is required for the induction of p53-dependent apoptosis.

a, b Western blot measuring the changes in the indicated protein after 48 h treatment with belinostat (PXD101; 0.1 μM) and cisplatin (CDDP; 10 μM) in Calu-1 isogenic cells generated through site-directed mutagenesis (p53^−/−, p53^wt, p53^R158G, p53^{R158G(DBD, K-A)}, p53^{R158G(CT, K-A)}, p53^R158G(K20A)) (n = 3 independent experiments) (a). β-actin shown as loading control. Densitometry of cleaved PARP (x-axis) and caspase 3 (y-axis) was quantified and normalized to β-actin, tabulated as ratio of belinostat/cisplatin combination to cisplatin alone (b). Data are represented as mean of three independent experiments. c, d Cell cycle analysis (PI staining) (c) and apoptotic profile (Annexin V staining) (d) were evaluated 48 hours post-treatment. Data are represented as mean IC₅₀ ± SD (n = 4 independent experiments). Two tailed Student’s t-test; *P < 0.05. e–i RT-qPCR analyses of the changes in mRNA levels of MDM2 (e), CDKN1A (f), PUMA (g), PMAIP1 (Noxa) (h), BAX (I) in p53^−/−, p53^wt, p53^R158G cells 48 h post-treatment. Data are represented as mean ± SD (n = 3 independent experiments). j Immunofluorescence staining was performed to determine the colocalization of acetyl-p53 (Lys382) (Alexa Fluor-488) and p-p53 (Ser15) (Alexa Fluor-568) in p53^wt and p53^R158G cells. Eight independent fields were taken for each condition with a minimum of 50 nuclei per field. Representative confocal images shown at ×40 magnification (n = 3 independent experiments). Merged images displayed with blue indicates DAPI, green indicates acetyl-p53, red indicates p-p53, whereas yellow indicates colocalization of p- and acetyl-p53. Scale bar, 50 µm. k Tenovin-6 IC₅₀ was quantified in Calu-1 isogenic clones 72 h post-treatment. Data are represented as mean IC₅₀ ± SD (n = 3 independent experiments). Two tailed Student’s t-test; *P < 0.05; **P < 0.01. l Western blot indicating changes in the indicated proteins in p53^−/−, p53^wt, p53^R158G cells 48 h after vehicle or tenovin-6 treatment (3 or 10 μM). β-actin shown as loading control (n = 3 independent experiments).