Fig. 3
Exosomal circZNF451 interacts with and enhances FXR1 ubiquitination in macrophages. A After coculturing with H1299 and A549 cells (5 × 105) for 48 h, the RIP assay was performed in macrophages (1 × 106; PMA stimulated THP-1 cells for 24 h) with IgG and AGO2 antibody. B After coculturing with A549 for 48 h, RNA pulldown assay was performed with the biotinylated circZNF451 probe to detect the interaction with FXR1 in macrophage. C After coculturing with A549 for 48 h, RIP assay was performed in macrophage to confirm the interaction between circZNF451 and FXR1 with the FXR1 antibody. D Colocalization of circZNF451 and FXR1 in macrophage was detected via FISH; scale bar, 50 μm. E After coculturing with A549/H1299-shcircZNF451 cells, FXR1 expression in macrophages (1 × 106) was detected via western blotting. F After coculturing with A549/H1299-shcircZNF451 and H1395/H1975-circZNF451 cells (5 × 105), FXR1 expression in macrophages (1 × 106) was detected via qRT-PCR. G Vectors encoding three fragments of FXR1 containing the three RNA-binding domains (KH1, KH2, and RGG box) with circZNF451 were transfected into 293 T cells, and RIP was performed with IgG and Flag antibody to detect circZNF451 enrichment. H Interaction between FXR1 and circZNF451 in macrophage was verified by RNA pulldown with biotinylated circZNF451 WT and mutant probes. I After administration of MG132 (20 μM) for 48 h, FXR1 expression in macrophages cocultured with H1395/H1975-circZNF451 was detected by western blotting. J IP was performed to measure FXR1 ubiquitination after coculturing macrophages with LUAD cell lines with silenced (A549 and H1299) or overexpressed (H1395 and H1975) circZNF451 and GW4869 administration (20 μM) for 48 h. A, C and G used the two-tailed, unpaired Student’s t test. F was analyzed by one-way ANOVA test after adjusting for multiple comparisons. All experiments with statistical analysis have been repeated for at least three times