Fig. 5
Contact with Interleukin-30 knockout PC cells downregulates 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 IL30KO-DU145 versus HUVEC, or HAEC, cocultured with wild type DU145. Results obtained from control NTgRNA-treated 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 IL30KO-PC3 versus HUVEC, or HAEC, cocultured with wild type PC3. Results obtained from control NTgRNA-treated 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 IL30KO-DU145 (yellow bars), or IL30KO-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 IL30KO-DU145 (red bars), or IL30KO-PC3 (blue bars) versus HAEC cocultured with wild type DU145, or PC3, respectively. Only genes that were regulated in both endothelial cell types by contact with both PC cell type are represented. A significant threshold of a twofold change in gene expression corresponded to p < 0.001. Experiments were performed in duplicate. D, E Immunohistochemistry showing that expression of TGFα was marked in wild type PC3 (D, a) and DU145 (E, a) and their vascular endothelial network (red arrows), whereas it was scanty in IL30KO-PC3 (D, b) and IL30KO-DU145 tumors (E, b) and almost absent in their vascular network. Results from NTgRNA-tumors were comparable to wild type tumors. Magnification: X400. F, G Western blot analysis of CXCL6 and THBS2 protein expression in ECs cocultured with IL30KO-DU145 (F) or IL30KO-PC3 (G) versus ECs cocultured with control (NTgRNA-treated) or wild type (WT) DU145 (F) or PC3 (G) cells. Representative images of experiments in triplicate. H Western blot analysis of IGF1 protein expression in ECs cocultured with IL30 overexpressing or knockout DU145 or PC3 cells. Representative images of experiments in triplicate