Fig. 8

Analysis of STAT4 activation and its downstream effects in PC3 and DU145 cells. A Western blot analysis showing phosphorylated STAT4 (pSTAT4) and total STAT4 levels in whole-cell lysates (WCL), cytoplasmic extracts (CE), and nuclear extracts (NE) of PC3 and DU145 cells treated with IL-11. Lamin B1 and β-Actin served as nuclear and cytoplasmic loading controls, respectively. B TRRUST database table indicating potential STAT4 target genes and their mode of regulation. C Immunofluorescence microscopy images depicting localization of STAT4 and pSTAT4 (red) in DU145 and PC3 cells, with and without IL-11 treatment. DAPI staining (blue) indicates nuclei. D Chromatin immunoprecipitation (ChIP) followed by qPCR analysis of c-MYC promoter regions in PC3 and DU145 cells. PCR products were visualized on an agarose gel to confirm the size of the amplicons (211 bp). E Quantitative representation of the fold enrichment of STAT4 binding to target gene promoters (IFNG, IL2RA, IRF1, c-MYC, NOX1, PIM1, IRF1, S100A4, SOCS3) in PC3 and DU145 cells, with and without IL-11 treatment. Data are presented as the mean ± SD of triplicate experiments; *p < 0.05, **p < 0.01 compared to IgG control. F Western blot analysis to evaluate the effect of STAT4 knockdown (Sh STAT4) on IL-11-induced pSTAT4 and c-MYC expression in PC3 and DU145 cells. GAPDH was used as a loading control. G Cell viability assay of PC3 cells with different treatments (control, OE IL-11, STAT4 knockdown, and combined OE IL-11 with STAT4 knockdown) across a range of docetaxel (DTX) concentrations. H Similar to (G) but showing the results for DU145 cells. The numbers indicate the IC50 values calculated for each treatment condition