Fig. 1

Constitutive expression of IL30 in human PC cells and IL30-dependent regulation of their proliferation, migration and invasion abilities. A, B Cytofluorimetric analyses of IL30 expression in human PC cells, DU145 (A) and PC3 (B). The DU145 cells showed a mean fluorescence intensity (MFI) ratio of 2.34, whereas the MFI ratio of PC3 cells was 1.95. The MFI was obtained calculating the ratio between the fluorescence of the samples and their isotype controls. Red lines: isotype control. Blue lines: anti-IL30 Abs. Results obtained from NTgRNA-treated cells were comparable with those from WT and EV-transfected cells. Experiments were performed in triplicate. C, D Western blot analyses of IL30 protein expression in the cytosolic and plasma membrane fractions of wild type, NTgRNA-treated, IL30KO, EV and IL30 gene-transfected DU145 (C) and PC3 (D) cells. E, F Cryo-immunoelectron microscopy of IL30 in DU145 (E) and PC3 (F) cells, showing IL30 localization, by gold particles, in WT (a), IL30KO (b) and IL30-overexpressing (c, d) cells. The gold particles were more frequent in IL30-overexpressing DU145 and PC3 cells (c, d) than in WT cells (a), whereas they were absent in IL30KO cells (b). In both DU145 and PC3 cells, the gold particles specifically delineated the plasma membranes (black arrows) and their microvilli-like structures (red arrows), the endoplasmic reticulum and associated cytoplasmic vesicles (green arrows). One out of four labeling experiments is shown. PM, plasma membrane; N, nucleus; er, endoplasmic reticulum; M, mitochondrion. Scale bars: 100 nm. G, H Cytofluorimetric analyses of gp130 (CD130) and IL6Rα (CD126) expression in DU145 (G) and PC3 (H) cells. Red lines: isotype control. Experiments were performed in triplicate. I, J MTT assay of DU145 (I) and PC3 (J) cells, after 48 h of treatment with anti-IL30 Abs (0.5–5.0 µg/mL). ANOVA: p < 0.0001. I *p < 0.01, Tukey HSD test compared with 0.0 µg/mL. **p < 0.05, Tukey HSD test compared with 0.0, 0.5 and 1.0 μg/mL. J *p < 0.01, Tukey HSD test compared with 0.0 and 0.5 µg/mL. **p < 0.01, Tukey HSD test compared with 0.0, 0.5 and 1.0 μg/mL. Results are expressed as mean ± SD. K, L MTT assay of IL30 gene-transfected, IL30-DU145 (K) and IL30-PC3 (L) cells, versus EV-transfected and WT cells. K ANOVA, p < 0.05. *p < 0.05, Tukey HSD test compared with WT and EV-transfected cells. L ANOVA, p < 0.0001. *p < 0.01, Tukey HSD test compared with WT and EV-transfected cells. Results are expressed as mean ± SD. M, N The treatment with anti-IL30 Abs (48 h) significantly decreased the number of DU145 and PC3 cells, that migrated (M) across the polycarbonate membrane insert or that invaded (N) the basement membrane matrix layer. By contrast, IL30 overexpression (IL30 over) significantly increased the number of migrating and invading DU145 (M) and PC3 (M) cells. Results obtained from EV-transfected cells are comparable with those from untreated wild-type cells (CTRL). Experiments were performed in triplicate. Results are expressed as mean ± SD. ANOVA, p < 0.01. *p < 0.05, Tukey HSD test compared with CTRL. **p < 0.05, Tukey HSD test compared with CTRL and cells treated with anti-IL30 Abs. O, P Quantitative western blot analysis of the expression of phospho-STAT1α and β isoforms, and phospho-STAT3α and β isoforms in DU145 (O) and PC3 (P) cells, and corresponding IL30 gene-transfected (IL30) cells, or IL30 gene knockout (IL30KO) cells, or wild-type cells treated with anti-IL30 Abs (IL30Abs). Expression of phospho-STAT1α was 15.93, and 53.26 times higher in IL30-DU145 and IL30-PC3, respectively, than in wild-type cells. Expression of phospho-STAT1β was 31.46, and 17.56 times higher in IL30-DU145 and IL30-PC3, respectively, than in wild-type cells. Expression of phospho-STAT3α and β was higher in IL30-DU145 (2.33 and 3.12 times) than in wild-type cells, whereas it was reduced in IL30KO-DU145 (− 6.63 and − 22.75 times) and in DU145 cells treated with anti-IL30 Abs (− 5.94 and − 23.89 times). Expression of phospho-STAT3α and β was higher in IL30-PC3 (2.22 and 4.17 times) than in wild-type cells, whereas it was reduced in IL30KO-PC3 (− 3.02 and − 6.42 times) and in PC3 cells treated with anti-IL30 Abs (− 2.96 and − 3.32 times). Results from control EV-transfected or NTgRNA-treated cells were comparable with those from wild-type cells. Q MTT assay of STAT1 siRNA- or STAT3 siRNA-transfected IL30-DU145 (a) and IL30-PC3 (b) cells. (a) ANOVA, p < 0.01; *p < 0.05, Tukey HSD test compared with DU145 cells; **p < 0.05, Tukey HSD test compared with IL30-DU145 cells. (b) ANOVA, p < 0.0001; *p < 0.01, Tukey HSD test compared with PC3 cells; **p < 0.01, Tukey HSD test compared with IL30-PC3 cells. Results from cells transfected with STAT1 or STAT3 scrambled siRNAs are comparable with those from IL30-overexpressing cells. Results are expressed as mean ± SD. R Migration assay of STAT1 siRNA- or STAT3 siRNA-transfected IL30-DU145 (a) and IL30-PC3 (b). ANOVA, p < 0.05. *p < 0.05, Tukey HSD test compared with DU145 (a) and PC3 (b) cells; **p < 0.05, Tukey HSD test compared with IL30-DU145 (a) and IL30-PC3 (b) cells. Results from cells transfected with STAT1 or STAT3 scrambled siRNAs are comparable with those from IL30-overexpressing cells. Results are expressed as mean ± SD. S Invasion assay of STAT1 siRNA- or STAT3 siRNA-transfected IL30-DU145 (a) and IL30-PC3 (b) cells. ANOVA, p < 0.0001. *p < 0.01, Tukey HSD test compared with DU145 (a) and PC3 (b) cells; **p < 0.01, Tukey HSD test compared with IL30-DU145 (a) and IL30-PC3 (b) cells. Results from cells transfected with STAT1 or STAT3 scrambled siRNAs are comparable with those from IL30-overexpressing cells. Results are expressed as mean ± SD