Fig. 3
Contact with Interleukin-30 overexpressing PC cells leads to kinase-phosphorylation of multiple signaling proteins and expression of inflammation and angiogenesis-related genes in endothelial cells. A Fold differences of mRNAs of angiogenesis-related genes between HUVEC (yellow bars) or HAEC (red bars) cocultured with IL30-DU145 versus HUVEC, or HAEC, cocultured with wild type DU145. Results obtained from control EV-transfected cells were comparable to those from wild type cells. A significant threshold of a twofold change in gene expression corresponded to p < 0.001. Experiments were performed in duplicate. B Fold differences of mRNAs of angiogenesis-related genes between HUVEC (yellow bars) or HAEC (red bars) cocultured with IL30-PC3 versus HUVEC, or HAEC, cocultured with wild type PC3. Results obtained from control EV-transfected cells were comparable to those from wild type cells. A significant threshold of a twofold change in gene expression corresponded to p < 0.001. Experiments were performed in duplicate. C Fold differences of mRNAs of angiogenesis-related genes between HUVEC cocultured with IL30-DU145 (yellow bars), or IL30-PC3 (green bars), versus HUVEC cocultured with wild type DU145, or PC3, respectively. Fold differences of mRNAs of angiogenesis-related genes of HAEC cocultured with IL30-DU145 (red bars), or IL30-PC3 (blue bars) versus HAEC cocultured with wild type DU145, or PC3, respectively. Genes regulated, in both endothelial cell types, by coculture with both PC cell lines are represented. A significant threshold of a twofold change in gene expression corresponded to p < 0.001. Experiments were performed in duplicate. D Cytofluorimetric analyses of ITGAV (at the top), and of JAG1 (at the bottom) expression in HUVEC and HAEC untreated (CTRL) or treated with rhIL30 (50 ng/ml for 24 h and restimulation with 100 ng/ml for 3 h). Experiments were performed in duplicate. E Fold differences of mRNAs of angiogenesis-related genes between HUVEC cocultured with IL30-DU145 versus HUVEC cocultured with DU145 (dark green bars), or HUVEC cocultured with IL30-PC3 versus HUVEC cocultured with PC3 (light green bars). Only genes up- or down-regulated in both coculture conditions are represented. A significant threshold of a twofold change in gene expression corresponded to p < 0.001. Experiments were performed in duplicate. F, G Representative images from n = 3 experiments of a phospho-kinase array (array of 43 kinases in duplicate spots) performed on HUVEC (F) and HAEC (G), after treatment with rhIL30 (50 ng/ml for 3 h and subsequent restimulation with 100 ng/ml for 15 min). Phosphorylation of signaling molecules (on the left side of the panels) is represented by paired spots, according to intensity of phosphorylation. Histograms (on the right side of the panels) represent the amounts of phosphorylated proteins, expressed as mean pixel density, measured by ImageJ software, of each pair of spots present on the stained membrane. *p < 0.05, Student's t-test compared with CTRL. H Venn diagram representing the signaling proteins, which are phosphorylated in HUVEC (light blue circle) and HAEC (pink circle) after treatment with rhIL30. Overlapping circles illustrate only the proteins phosphorylated after rhIL30 treatment in both EC types