Fig. 1

Interleukin-30 overexpression in PC cells stimulates endothelial cell proliferation and promotes vascular tube formation. A Cytofluorimetric analyses of IL30 (a, b), CD130 (c, d) and CD126 (e, f) expression in HUVEC top of the panel, and HAEC, bottom of the panel. Both EC types did not express IL30 but expressed the gp130 (CD130) and IL6Rα (CD126) receptor chains. Red lines: isotype control. Experiments were performed in triplicate. B Cytofluorimetric analyses of EC proliferation by Ki67 staining. When compared to ECs cultured alone (a, b), ECs cocultured with PC cells, DU145 (c, d), showed higher proliferation. Endothelial proliferation was suppressed by coculture with IL30KO-DU145 cells (e, f) and enhanced by coculture with IL30-DU145 cells (g, h). Red lines: isotype control. Experiments were performed in triplicate. C, D MTT assay of HUVEC (C) and HAEC (D), after 48 h of treatment with rhIL30 (50 ng/ml). *p < 0.0001, Student's t-test compared with untreated cells. Results are expressed as mean ± SD. Experiments were performed in triplicate. E Cytofluorimetric analyses of HUVEC (top of panel) and HAEC (bottom of panel) after the treatment with rhIL30 (50 ng/ml overnight and restimulation with 100 ng/ml for 4 h) revealed the upregulation of VCAM-1 (a-d) and P-selectin (e–h) compared to untreated (CTRL) cells. Experiments were performed in triplicate. F Confocal microscopy images of ECs (stained red with anti-CD31 Abs) cocultures with IL30-DU145 (a-d), wild type DU145 (e–h), or IL30KO-DU145 (i-l) cancer cells (stained green with anti-EpCAM Abs). Nuclei stained blue with DAPI. Representative images from three experiments. Magnification: X400. G Histograms representing the automated quantitative analysis, performed on confocal microscopy images, of the mean number ± SD of CD31⁺ECs after coculture with wild type, IL30-overexpressing or IL30 knockout DU145 (G) or PC3 (H) cells. Results are expressed as mean ± SD of CD31⁺cells per field. ANOVA: p < 0.0001. *p < 0.01, Tukey HSD Test compared with ECs + DU145 or PC3. **p < 0.01, Tukey HSD Test compared with ECs + DU145 or PC3 and ECs + IL30-DU145 or IL30-PC3. I, J Confocal microscopy images of capillary tube formation in Matrigel by red stained (anti-CD31 Abs) HUVEC (I) and HAEC (J) untreated (CTRL, a-c) or treated (50 ng/ml for 24 h, d-f) with rhIL30. Nuclei stained blue with DAPI. Representative images of three experiments. Magnification: X400. K, L Histograms representing the automated quantitative analysis, performed on confocal microscopy images, of the mean number ± SD of CD31⁺HUVEC (F) or CD31⁺HAEC (G) capillaries per field (X400), developed in Matrigel with or without (CTRL) addition of rhIL30. *p < 0.0001, Student's t-test versus CTRL. Experiments were performed in triplicate