Fig 1: Principal component analysis, Pearson's correlogram and unsupervised hierarchical clustering analysis of untreated, IFN-γ-or IL-27-treated SKOV3 cellsA. Two-dimensional scatter plot of the principal component analysis of SKOV3 Untreated (blue), IFN-γ- (orange) and IL-27-treated (red dots) samples.B. The Pearson's correlogram depicts the coefficient values in a pseudo-color scale, which extends from 0.1 (light blue) to 0.9 (red). The dendrogram displays the results of an unsupervised hierarchical-clustering analysis placing similar Pearson's coefficient values near each other. All the samples cluster according to treatment.C. Unsupervised hierarchical-clustered heatmap of 990 proteins identified by Multiple-samples test ANOVA performed on the SKOV3 cell line. The amount of each protein in individual samples is represented by the color scheme in which red and blue indicate high and low expression of proteins, respectively. Three independent biological replicates of cells treated with IL-27 or IFN-γ or left untreated (CTR) are shown. Proteins are clustered into 6 groups according to their expression value. Cluster #3 and cluster #6 represent a concordant modulation by IFN-γ and IL-27 treatments. In particular, 489 were up-regulated in cluster #6 and 325 were down-regulated in cluster #3 by both cytokines. A smaller number of proteins (176) was differentially modulated by the two cytokines and clustered into 4 small groups, relative to untreated cells.
Fig 2: IL-27 increases HLA class I membrane expression in human ovarian cancer cells in vitroFACS analysis of surface HLA class I expression in five ovarian cancer cell lines and one primary culture (IST-A161, passage 15) of serous ovarian carcinoma cells from ascites, cultured in the presence of medium (baseline), IL-27 or IFN-γ (induced). Isotype-matched unrelated Ig staining control is indicated (ctrIg). Numbers in brackets are Median Fluorescence Intensity (MFI) values calculated as median HLA class I (W6/32 mAb) minus median Ig control.
Fig 3: IL-27 increases HLA class I membrane expression in human NSCLC and NB cells in vitro.FACS analysis of surface HLA class I expression in three NSCLC A. and three neuroblastoma B. cell lines, cultured in the presence of medium (baseline), IL-27 or IFN-γ (induced). Isotype-matched unrelated Ig staining control is indicated (ctrIg). Numbers in brackets are Median Fluorescence Intensity (MFI) values calculated as median HLA class I (W6/32 mAb) minus median Ig control.
Fig 4: IL-27 predominantly mediates STAT1 phosphorylation in MM cell lines. Western blot analysis of tyrosine phosphorylated (P)-STAT1, P-STAT3, total STAT1 and total STAT3 proteins (A) in MPP89 cells stimulated for 20 min with medium (CTR) or the indicated cytokines of the IL-12 family and (B) in MSTO, MPP89, and IST-MES1 cells stimulated for 20 min with medium (CTR), IL-6, sIL-6R/IL-6 chimera, and IL-27. Total STATs and α-tubulin were used as loading controls.
Fig 5: Volcano plot representation of differentially expressed proteinsPlots represent untreated (CTR) and IL-27-treated A., CTR and INF-γ-treated B. and IL-27 and IFN-γ-treated C. SKOV3 cell samples. Red dots represent proteins that display both large-magnitude fold-changes (x-axis, to the right there are proteins up-regulated after treatment) as well as high statistical significance (−log10 of P value, y-axis). The black line shows where FDR = 0.05 and s0 = 0.5. Gray dots represent proteins whose fold-change is < 2 (log2 = 1) or the P > 0.05. Proteins labeled with gene name (inset) are the most modulated ones.
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