Fig. 8

CircKEAP1 provokes cellular immune responses via RIG-I. (A). RNA sequencing was conducted in control and circKEAP1 overexpression cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway analyses were performed to find the most differentially-changed signaling pathways. (B). GSEA analysis for circKEAP1 compared to the control group. (C). Fold change of the indicated mRNAs in circKEAP1 knockdown and control OS cells was measured by qRT-PCR. (D). Expression levels of the indicated mRNAs in cells following RIG-I knockdown and/or circKEAP1-overexpression in U2OS and HOS cell lines. (E). Protein levels in HOS and U2OS cells with circKEAP1 overexpression and/or RIG-I knockdown were detected by western blotting. (F). Levels of CXCL10, CCL5 and IFNγ in the supernatants of cells was measured by ELISA following RIG-I knockdown or/and circKEAP1 overexpression in U2OS and HOS cell lines. (G). Expression of inflammatory cytokines in the supernatants of cells was characterized using a human inflammation antibody array. (H). Protein expression of STAT1 and P65 was detected by western blotting in the nuclear and cytoplasmic fractions of the indicated cells. (I). Cellular localization of STAT1 and P65 was detected by immunofluorescence staining (scale bars, 20 μm). (J). Biotin pulldown assay was performed using circKEAP1 probe and the anti-Flag was detected in U2OS and HOS cells. (K). FISH-IF staining was performed to show the cellular localization of circKEAP1 and RIG-I (scale bars, 20 μm). Error bars represent three independent experiments. *, **, *** indicates significant differences compared with the control or indicated group at a p value < 0.05, < 0.01, < 0.001, respectively