Fig 2: Both mutant p53 and KLF5 increase PLA2G16 expression in pancreatic cancer. A, A heatmap showing TP53 mutation and PLA2G16 expression in pancreatic adenocarcinoma (PAAD) cases in The Cancer Genome Atlas (TCGA). B, A violin plot chart comparing PLA2G16expression between PAAD cases with or without TP53 mutations. C, Flow chart showing the screening process to identify PLA2G16 correlated TFs in TP53 mutant PAAD cases. D and E, Comparison of KLF5 mRNA expression between normal pancreas in GTEx and PAAD in TCGA (D) and between PAAD cases with or without TP53 mutations (E). F, Representative images of KLF5 protein expression in PAAD tissue. Image credit: Human Protein Atlas, from: https://www.proteinatlas.org/ENSG00000102554-KLF5/pathology/pancreatic+cancer. G and H, Plot charts showing the correlation between PLA2G16 and KLF5 mRNA expression in TP53 mutant (G) and TP53 wild-type (H) PAAD cases. I and J, K-M survival analysis of PFS (I) and DSS (J) in PAAD cases in TCGA Pan-Cancer, by median KLF5 expression separation. K and L, Quantitative real-time PCR (qRT-PCR) analysis of PLA2G16 mRNA expression in PANC-1 and MIA-PaCa-2 cells 48 h after lentiviral-mediated TP53 (K) or KLF5 (L) inhibition. M, Western blot analysis of p53, KLF5 and PLA2G16 protein expression 72 h after lentiviral-mediated TP53 (up) or KLF5 (down) inhibition. N and O, qRT-PCR (M) and Western blot assay (O) of PLA2G16 expression in PANC-1 and MIA-PaCa-2 cells with TP53 and KLF5 inhibition (shTP53#2 and shKLF5#1) separately or in combination

Fig 3: Mutant p53 enhances KLF5-induced PLA2G16 transcriptional activation. A, Predicted binding sites of KLF5 in the promoter region of PLA2G16. B, The promoter activity of the PLA2G16 gene was measured using a dual-luciferase reporter assay. PANC-1 and MIA-PaCa-2 cells were transfected with pGL3-basic or reporter constructs carrying different lengths of the 5'-flanking region of the PLA2G16 promoter as indicated. C-E, KLF5 depletion reduced the activity of the PLA2G16 promoter. PANC-1 and MIA-PaCa-2 cells with or without lentiviral-mediated KLF5 inhibition were transfected reporter constructs carrying pGL3-(-1303/+239) (C), pGL3-(-400/+239) (D) and pGL3-(-100/+239) (E). 24 h later, luciferase activity was determined. F, Western blot assay of KLF5 expression 48 h after lentiviral-mediated overexpression. G-J, 24 h after infection with lenti-KLF5 or vector, PANC-1 and MIA-PaCa-2 cells were transfected reporter constructs carrying pGL3-(-1303/+239) (G-H) or mutant-pGL3-(-1303/+239) with mutant sequences (C to A) of the four binding sites (I, J). 24 h later, luciferase activity was determined. K, Schematic image showing the location of the designed primer sets for ChIP-quantitative polymerase chain reaction (qPCR) assay, by anti-KLF5 immunoprecipitation. L-O, ChIP-qPCR assays were performed using anti-KLF5 (L, M) or anti-TP53 (N, O) and control IgG antibodies in PANC-1 (L, N) and MIA-PaCa-2 (M, O) cells. Fold enrichment of the indicated PLA2G16 promoter segments was calculated. P, Co-IP assay to explore the potential binding between KLF5 and p53 in MIA-PaCa-2 and PANC-1 cells. Q and R, ChIP-qPCR assays were performed using anti-KLF5 in MIA-PaCa-2 (Q) and PANC-1 (R) cells with or without TP53 inhibition. Fold enrichment of the indicated regions of the PLA2G16 promoter was calculated. *P < .05; **P < .01; ***P < .001

Fig 4: PLA2G16 up-regulation was also associated with gene-level copy amplification and hypomethylation. A, A heatmap showing PLA2G16 expression, copy number alteration and methylation level of nine 5’-cytosine-phosphodiester bond-guanine-3’ sites in pancreatic adenocarcinoma (PAAD) cases in The Cancer Genome Atlas (TCGA). B, A plot chart showing the correlation between PLA2G16 expression and its copy number in 176 PAAD cases in TCGA. C, Comparison of PLA2G16 expression copy number between TP53 wild-type and mutant PAAD cases. D, A plot chart showing the correlation between PLA2G16 expression and the ß-value of cg09518969 in 177 PAAD cases in TCGA. E, Comparison of cg09518969 methylation value between TP53 wild-type and mutant PAAD cases

Fig 5: X15695-mediated regulation of AR action in prostate cancer cells. A, Western blot analysis of BAG1 in LNCaP cells transduced with BAG-1 shRNA and control shRNA. Antibodies to vinculin, AR, tubulin, and GAPDH were used as controls. B, Quantification of the effect of X15695 on clonal expansion of control and BAG1 shRNA knockdown LNCaP cells. Each point represents the mean ± SEM. n = 3. C, Western blot analysis of AR and BAG1 in extracts of LNCaP and 22Rv.1 cells treated with the indicated concentrations of X15695 for 24 hours. ß-actin was used as a loading control. D–F, GSEA plots of the topmost signaling pathways and heat maps of log2 fold-change in gene expression in the comparison of DHT + X15695 versus DHT and X15695 versus vehicle in LNCaP cells. G, Quantitative RT-PCR was carried to detect the effect of X15695 on DHT- mediated expression of the indicated target genes in LNCaP cells. Cells were treated with vehicle or 10 nmol/L DHT and X15695 (5 µmol/L) for 16 hours. The results are the means ± SEM (n = 4; *, P = 0.05; ***, P = 0.001; ****, P = 0.0001; ns, not significant). H, 22Rv.1, LAPC-4, and LNCaP cells were transfected with control and p53 siRNA for 16 hours and treated with vehicle or X15695 (5 µmol/L) for 24 hours. Western blot analysis was performed with anti-p53 and anti-ß-actin antibodies. I, MTT cell viability assay carried out with 22Rv.1, LAPC-4, and LNCaP cells previously transfected with control and p53 siRNA and treated with vehicle and X15695 for 72 hours. The results are the mean ± SEM (n = 3; *, P = 0.05).