Fig. 5: p53 transactivates Panx1 to drive IL-6 induction.

A, B qRT-PCR analysis of IL-6 gene expression in control (siCtrl) and p53-silenced (sip53) A549 cells untreated or treated with (A) 20 µM SA or (B) 0.5 µM Dox for 24 h. C The concentration of IL-6 present in the culture supernatants of siCtrl and sip53 A549 cells untreated or treated with 20 µM SA or 0.5 µM Dox for 24 h was determined by ELISA. D Western blot analysis of p53, phosphorylated STAT3 (Y705), and total STAT3 protein levels in siCtrl and sip53 A549 cells untreated or treated with 20 µM SA for 24 h. E, F qRT-PCR analysis of (E) p53 and (F) IL-6 gene expression in control (Ctrl) and p53-overexpressing (p53) A549 cells untreated or treated with 20 µM SA for 24 h. G The concentration of IL-6 present in the culture supernatants (upper panel) or the p53 protein levels (lower panel) of A549 cells untreated or treated with 0.5 µM Dox in the presence of increasing concentrations of the MDM2 inhibitor Nutlin-3 for 24 h was determined by ELISA or western blot, respectively. H qRT-PCR analysis of IL-6 gene expression in A549 cells untreated or treated with 0.5 µM Dox in the presence of increasing concentrations of Nutlin-3 for 24 h. I Schematic diagram of the human PANX1 gene locus with six potential p53 binding regions, −1911 to −1768 (ChIP 1), −1441 to −1329 (ChIP 2), −1277 to −1169 (ChIP 3), −817 to −652 (ChIP 4), −622 to −488 (ChIP 5), and −218 to −119 (ChIP 6), on the PANX1 promoter (upstream of TSS) and a 3′-untranslated region (ChIP 3′-UTR, +2675 to +2791, downstream of TSS), identified with the JASPAR program [46]. TSS, transcription start site. J Chromatin immunoprecipitation and quantitative real-time PCR (ChIP-qPCR) analysis of the relative enrichment of p53 at the indicated PANX1 promoter regions in A549 cells with or without 0.5 µM Dox treatment for 24 h. The relative enrichments of p53 at the CDKN1A (p21) promoter region (−2292 to −2169, upstream of TSS) and the 3′-untranslated (3′-UTR) region of the PANX1 gene were measured as positive and negative controls, respectively. K Panx1 gene expression in siCtrl and sip53 A549 cells untreated or treated with 20 µM SA or 0.5 µM Dox for 24 h was measured by qRT-PCR. L Western blot analysis of p53, Panx1, phosphorylated Akt (S473 and T308), total Akt, phosphorylated p65 NF-ĸB (S276), total p65 NF-ĸB, and γH2A.X protein levels in siCtrl and sip53 A549 cells untreated or treated with 20 µM SA for 24 h. M Dose-response curves showing the survival of siCtrl and sip53 A549 cells in response to increasing concentrations of SA treatment for 24 h. N The IC₅₀ values of SA against siCtrl and sip53 A549 cells were calculated from the nonlinear regression curves in Fig. 5M. Cell viability was measured by MTT assays. O, P The mRNA levels of the anti-apoptotic genes (O) Bcl-xL and (P) Mcl-1 in siCtrl and sip53 A549 cells untreated or treated with 20 µM SA for 24 h were measured by qRT-PCR. Error bars represent mean ± SD, n = 3. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test (A–C, E–H, K, O, P) or unpaired two-tailed Student’s t test (J, N). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns not significant. The full length uncropped original western blots related to this figure are provided in the Supplemental Material file.