Fig. 2

IL30-dependent regulation of PC driver genes in murine and human PC cells. A Mouse Prostate Cancer PCR Array. Fold differences of the mRNAs of PC driver genes between rmIL30-treated (red bars) and untreated wild-type murine (m) PC-SLCs, and between IL30KO-mPC-SLCs (blue bars) and mPC-SLCs. Results obtained from control NTgRNA-treated and untreated mPC-SLCs were comparable to those from wild type cells. A significant threshold of twofold change in gene expression corresponded to p < 0.001. Only genes with a fold change > 2, in at least one condition, up- or downregulation, are shown. Experiments were performed in duplicate. Stripped boxes represent scale breaks. The dashed lines represent the twofold change cutoff. B Cytofluorimetric analyses of gp130 (CD130) and IL6Rα (CD126) expression in TRAMP-C1 cells. Red lines: isotype control. Experiments were performed in triplicate. C MTT assay of TRAMP-C1 cells after 48 h treatment with rmIL30 at concentrations of 10, 35, 50 and 100 ng/mL. ANOVA: p < 0.0001. *p < 0.05, Tukey HSD test compared with 0 ng/mL. **p < 0.01, Tukey HSD test compared with 0, 10, 35 and 100 ng/mL. Results are expressed as mean ± SD. D, E The treatment with rmIL30 (6 h), significantly increased the number of TRAMP-C1 cells, which migrated (D) across the polycarbonate membrane insert, or which invaded (E) the basement membrane matrix layer. Results are expressed as mean ± SD. *Student’s t test: p = 0.0001 (D), p = 0.00001 (E), compared with untreated (CTRL) cells. Experiments were performed in triplicate. F Mouse Prostate Cancer PCR Array. Fold differences of the mRNAs of PC driver genes between rmIL30-treated (red bars) and untreated wild-type TRAMP-C1 cells. A significant threshold of a twofold change in gene expression corresponded to p < 0.001. Only genes with a fold change > 2 are shown. Experiments were performed in duplicate. Stripped boxes represent scale breaks. The dashed lines represent the twofold change cutoff. G Venn diagram representing the “PC Driver Genes” which are up- and/or downregulated by IL30 (treatment with rmIL30 or human IL30 gene knockout) in TRAMP-C1 (purple circle), PIN-SC (red circle), DU145 (green circle) and PC3 cells (blue circle). Overlapping circles illustrate the sharing of IL30-regulated genes between different cell lines. H Human Prostate Cancer PCR Array. Fold differences of mRNAs of PC driver genes between IL30-overexpressing IL30-DU145 cells (red bars) and wild-type DU145 cells, and between IL30KO-DU145 cells (blue bars) and wild-type cells. Results obtained from control NTgRNA-treated and EV-transfected DU145 cells were comparable to those from wild-type cells. A significant threshold of twofold change in gene expression corresponded to p < 0.001. Only genes with a fold change > 2, in at least one condition, up- or downregulation, are shown. Experiments were performed in duplicate. The dashed lines represent the twofold change cutoff. I Fold differences of the mRNAs of PC driver genes between IL30-overexpressing IL30-PC3 cells (red bars) and wild-type PC3 cells, and between IL30KO-PC3 cells (blue bars) and wild-type cells. Results obtained from control NTgRNA-treated and EV-transfected PC3 cells were comparable to those from wild-type cells. A significant threshold of a twofold change in gene expression corresponded to p < 0.001. Only genes with a fold change > 2, in at least one condition, up- or downregulation, are shown. Experiments were performed in duplicate. The stripped box represents a scale break. The dashed lines represent the twofold change cutoff. J Western blot analysis of BCL2 protein expression in WT, EV- or IL30 gene-transfected, NTgRNA-treated and IL30KO-PC3 cells, and in WT, NTgRNA-treated and IL30KO-DU145 cells. K Western blot analysis of NFKB1 protein expression in EV- or IL30 gene-transfected, NTgRNA-treated and IL30KO, PC3 and DU145 cells. Results obtained from control NTgRNA-treated and EV-transfected cells were comparable to those from wild-type cells. L Western blot analysis of DKK3 and SOCS3 protein expression in WT, EV- or IL30 gene-transfected, NTgRNA-treated and IL30KO-PC3 cells, and in WT, NTgRNA-treated and IL30KO-DU145 cells