Additional file 1 of Interleukin-30 subverts prostate cancer-endothelium crosstalk by fostering angiogenesis and activating immunoregulatory and oncogenic signaling pathways
Creators: name: Ciummo, Stefania Livia
name: Sorrentino, Carlo
name: Fieni, Cristiano
name: Di Carlo, Emma
Publisher: name: figshare
Year: 2023
Subjects: subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Plant Biology
subject: Computational Biology

description: Additional file 1: Supplementary Fig. S1. Cytofluorimetric analyses of endothelial cell marker expression in HUVEC (top of the panel), and HAEC (bottom of the panel). Both endothelial cell types expressed CD31/PECAM-1, CD34, CD54, CD309/VEGFR2 and vWF, but did not express CD45. Red lines: isotype control. Experiments were performed in triplicate. Supplementary Fig. S2. Western blot analysis showing that both HUVEC and HAEC express EBI3, but not the p28 (IL30) subunit of the heterodimeric (p28/EBI3) cytokine IL27. A representative image of triplicate experiments is shown. Supplementary Fig. S3. Cytofluorimetric analysis of cell apoptosis, by annexin V staining, in HUVEC (top of the panel) and HAEC (bottom of the panel) co-cultured or not with wild type DU145, IL30KO-DU145 or IL30-DU145. The negative annexin V staining indicates the absence of cells undergoing apoptosis in all conditions. Experiments were performed in triplicate. Supplementary Fig. S4. Western blot analysis of ANG, CXCL9, EDN1 and TGFB2 protein expression in HAEC co-cultured with DU145 (left side of the panel) or PC3 cells (right side of the panel). Representative images of three experiments. Supplementary Fig. S5. Western blot analysis of phosphorylated HSP60, p53, CREB and GSK3b proteins in ECs (HUVEC and HAEC) treated with rhIL30. Supplementary Fig. S6. Expression of CXCR3 isoforms in HAEC and HUVEC, as determined by RT-PCR. CXCR3A: 111 bp; CXCR3B: 79 bp; CXCR3-alt: 135 bp. Supplementary Fig. S7. Western blot analysis of CXCL6 and THBS2 protein expression in ECs (HAEC) co-cultured with IL30KO-DU145 (A) or IL30KO-PC3 (B) versus ECs co-cultured with control (NTgRNA-treated) or wild type (WT) DU145 or PC3 cells. Western blot analysis of IGF1 protein expression in ECs co-cultured with IL30 overexpressing or knockout DU145 or PC3 cells (C). Representative images of experiments in triplicate. Supplementary Fig. S8. Immunohistochemical analyses of PC3 (A) and DU145 (B) tumors show that expression of LGALS4 (a, b) and TNFa (c, d) is stronger in IL30 overexpressing tumors when compared to the respective wild type tumors (e, f). Results from EV-tumors were comparable to wild type tumors. Magnification: X400. Supplemental Fig. S9. Expression of CXCR5 in PC tissues obtained from patients bearing IL30Neg PC or IL30Pos PC. Inset shows a magnification of CXCR5 positive tumor cells. Representative images of the immunohistochemical study are shown. Magnification: X400. Supplementary Table S1. Antibodies used in flow cytometry. Supplementary Table S2. List of genes included in the apoptotic signaling pathway. Supplementary Table S3. Antibodies used in immunostaining. Supplementary Table S4. Gene list of the RT2 Profiler Human Angiogenesis PCR Array (#PAHS-024ZR). Supplementary Table S5. Immunohistochemical analysis of IL30 expression, microvessel density and proliferation index in wild type, IL30 gene transfected or deleted tumors and control EV-transfected or NTgRNA-treated tumors. Supplementary Table S6. Immunohistochemical analysis of inflammation and immunity genes in wild type and IL30 gene knockout tumors. Supplementary Table S7. Gene list of the RT2 Profiler Human Prostate Cancer PCR Array (#PAHS-135ZR). Supplementary Table S8. Gene list of the RT2 Profiler Human Cancer Inflammation &amp; Immunity Crosstalk PCR Array (#PAHS-181Z). Supplementary Table S9. Immunohistochemical analysis of inflammation and immunity genes and prostate cancer driver genes in wild type and IL30 gene transfected tumors. Supplementary Table S10. Morphometric evaluation of IL30 and immunoregulatory and prostate cancer driver gene expression in prostate cancer samples from patients of the validation cohort*.