Fig 1: RNF213 knockdown downregulates DDAH1 and CYR61 at the transcript level. HeLa cells were treated with IFN-β in combination with knockdown of RNF213 as described in Figure 1 . (A) Immunoblotting on the actual samples used for proteome analysis confirmed efficient knockdown of RNF213 and interferon-induced upregulation of free and conjugated ISG15. Immunoblots against DDAH1, CYR61 and MVP validated downregulation of DDAH1 and CYR61, and upregulation of MVP upon knockdown of RNF213. Tubulin was used as loading control. Densitometry and statistical analysis on two more biological repeats confirmed significant up- or downregulation of the immunoblotting bands ( Supplementary Figure 3 ). (B) Real time qPCR upon knockdown of RNF213 showed significant upregulation of MVP in the presence of IFN-β and significant downregulation of CYR61 and DDAH1 transcripts in the presence and absence of IFN-β. Expression was normalized to housekeeping genes and shown relative to control (siScramble) populations set to 1. Data represents 3 biological replicates with 4 technical replicates per condition. (AVG ± SEM, two-tailed unpaired t-test (n = 3), *p < 0.05 and ***p < 0.001).
Fig 2: Enrichment of VM-related genes by EBV infection. a Venn diagram illustrating the overlap of differentially expressed genes (DEGs) in paired EBV-negative and EBV-positive NPC cells (CNE2 vs. CNE2-EBV and TW03 vs. TW03-EBV) by RNA-seq. b Bar plot ranking of the top enrichment score (p-value) values for the significant enrichment pathways according to KEGG analysis of DEGs between EBV− and EBV+ NPC cells. Only pathways with p < 0.05 are shown. c GO tree obtained from BiNGO showing the hierarchy of biological processes related to endothelial cell development, endothelial cell migration and angiogenesis between the EBV-positive and EBV-negative NPC cell pairs (padj < 0.05; see the color bar). The white nodes (padj > 0.05) were added to show the relationships among their downstream nodes. d Heatmap depicting FPKM values for vascular development and tube morphogenesis-related genes in EBV− and EBV+ NPC cell pairs and EBV+ cells with EBNA1 deletion by gRNAs. padj < 0.05. Of note, 36 out of 39 of these genes contain HRE motif in their promoter region (Supplementary Fig. 3) with the exception of three genes colored in green. e The average FPKM values of the DEGs in d. ns: not significant (one-tailed Wilcoxon matched pairs test). f (left) Serial sections of EBV-negative and EBV-positive NPC tissues were stained with H&E, EBER and antibodies targeting F3, CYR61, EphA2 and TNFRSF12A. Scale bars = 500 μm. (right) IHC scores of the indicated genes in 10 EBV-positive and 7 EBV-negative NPC biopsies. Means ± SD, **p < 0.01, two-tailed Mann–Whitney test
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