Supplementary Figure S5 from The IL-18 Antagonist IL-18–Binding Protein Is Produced in the Human Ovarian Cancer Microenvironment

Abstract

Supplementary Figure S5 - PDF file 470K, Biological effects of IL-27 on cells isolated from the ascites and on EOC cell lines. A-C: Involvement of IL-27 in IL-18BP expression in EOC cells. IL-18BP secretion is detected by ELISA in culture supernatants following A2774 and SKOV3 EOC cell stimulation with IL-27 (10 to 100 ng/ml for 48 h). The effect of IFN-gamma is shown for comparison (A). IL-27 stimulation (48 hours, at the indicated doses) mediates IL-18BP secretion by A2780 and OVCAR5 EOC cell lines, as detected by ELISA (B). ** P<0.005 at all dose levels versus untreated control. IL-27 up-regulates IL18BP mRNA expression in A2774 and SKOV3 cell lines, as assessed by RT-qPCR (C). D: A 10 minutes treatment with IL-27 (10 ng/ml) induces STAT1 tyrosine phosphorylation in A2774 and SKOV3 cells, as detected by Western blot analysis on cell lysates. Tubulin or beta-actin was used as loading controls. Numbers indicate molecular weight markers in kDa. E: Immunofluorescence and confocal microscopy analysis of constitive and IL-27-induced STAT1 tyrosine phosphorylation in EpCAM-positive and EpCAM-negative cells isolated from EOC ascites (specimen A98). Immunofluorescence was performed incubating 5x105 EOC cells with FITC-labeled anti-EpCAM antibody (Miltenyi). Cells, fixed and permeabilzed with BD Cytofix/Cytoperm solution (BD Pharmingen) were then incubated with rabbit anti-IL-18BP antibody (Epitomics) followed by DyLight 549-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories). PermaFluor (Thermo Scientific)-mounted slides were analyzed by confocal fluorescence microscopy using an Olympus (Olympus Optical) laser-scanning microscope FV500 equipped with an Olympus IX81 inverted microscope. Digital images were acquired through PLAPO 60x objectives, with the Fluoview 4.3b software program. Images were acquired sequentially as single trans-cellular optical sections, archived in TIFF and mounted using Photoshop. Merged images are shown. Bar=50micron. Arrows indicate EpCAM-positive pSTAT1-positive ascites cells, arrowheads indicate EpCAM-negative pSTAT1-positive ascites cells</p