Fig. 6

Degradation of FXR1 activates the ELF4–IRF4 pathway in macrophages. A Volcano map of mRNA expression in macrophages (PMA stimulated THP-1 cells for 24 h) cultured with H1395-circZNF451 and control cells. B GO and KEGG analysis of the differently expressed candidates (FC > 2, P < 0.05) that may be regulated by circZNF451 in macrophages. C Overlap of immune-related candidates in the differently expressed genes (n = 65) identified by RNA-seq and the predicted candidates for FXR1 in ENCORI (n = 10,182). D Dual immunofluorescence staining of ELF4 and CD163 in the TMA containing the tumor tissues of 113 LUAD patients and the correlation of ELF4⁺CD163⁺ macrophages with circZNF451 expression was explored by Spearman correlation analysis; scale bar, 100 μm. E Silencing of ELF4 in macrophages was verified by western blotting. F After coculturing with H1395-circZNF451 for 48 h, the influence of ELF4 silencing in LPS-stimulated macrophages on the M2 phenotype was confirmed by flow cytometry. G After coculturing with H1395-circZNF451 for 48 h, the M2 phenotype markers Arg1 and IL-10 were measured by qRT-PCR after ELF4 knockdown in macrophages stimulated by LPS. H The binding motif of ELF4 and three putative binding sites in the promoter region of IRF4. I A ChIP assay was performed in macrophage to verify the binding region of ELF4 in IRF4. J A luciferase assay was performed to confirm the binding of ELF4 in the three putative regions of IRF4. K IRF4 expression was detected in LPS-stimulated macrophage-siELF4 cells via western blotting after coculturing with H1395-circZNF451 cells for 48 h. L IRF4 expression in LPS-stimulated macrophages was measured by western blotting after coculturing with LUAD cell lines with silencing (A549 and H1299) or overexpression (H1395 and H1975) of circZNF451 and administration of GW4869 (20 μM) for 48 h. M The M2 phenotype of LPS-stimulated macrophage-siIRF4 cells was detected by flow cytometry after coculturing with H1395-circZNF451 cells for 48 h. F, G and J were analyzed by one-way ANOVA test after adjusting for multiple comparisons. I and M used the two-tailed, Student’s t test. All experiments with statistical analysis have been repeated for at least three times