Fig. 2

YAP promotes the M2 polarization of macrophages and repair of intestinal epithelial cells after inflammatory injury. (A) After THP-1 monocytes were stimulated with 100 ng/ml PMA for 24 h to obtain nonpolarized M0 macrophages, M1 macrophages were obtained by applying 100 ng/ml LPS and 20 ng/ml IFN-γ for 48 h, and the expression of macrophage markers and inflammatory factors was detected by western blotting and qPCR. (B) After nonpolarized M0 macrophages were obtained, M2 macrophages were induced with 20 ng/ml IL-4 for 48 h. Western blotting and qPCR were performed to detect the expression of macrophage markers and inflammatory factors. (C) Stable lentiviral transfection was used to construct YAP-overexpressing and empty control THP-1 cells, and 100 ng/ml PMA was incubated with the cells for 24 h to obtain YAP-overexpressing macrophages and control macrophages. Western blotting and qPCR were used to detect the expression of M1 and M2 macrophage markers and inflammatory factors. (D) After PMA-induced macrophages were generated, YAP expression in the macrophages was knocked down by transient siRNA transfection, and the corresponding target protein and mRNA expression levels were detected. (E) FHC cells were stimulated with 1 µg/ml lipopolysaccharide (LPS) for 24 h to establish an inflammatory intestinal epithelial cell model. The expression of the proinflammatory cytokines IL-6 and IL-1β and the anti-inflammatory cytokines IL-10 and IL-13 was detected by qPCR. (F) After LPS prestimulation, normal colonic epithelial cells (FHC cells) were cocultured with YAP WT macrophages or vector-transfected macrophages for 48 h, and the expression of YAP, the proliferation-associated factors PCNA and cyclin D1, and the apoptosis-associated factors caspase3 and caspase4 was detected by western blotting. (G-H) CCK-8 and scratch wound healing assays were performed to detect intestinal epithelial cell proliferation and migration after coculture. Asterisks indicate statistical significance, * p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001; ns, not significant; t test.