Fig. 5

TAMs polarized in OCM/AS via inactivating RhoA-YAP1 signaling cascade. (A) M0 MФs (THP-1) were stimulated with LPS/LFN-γ to make M1 MФs. After that, M1 MФs were treated with OCM or a control medium. RNA-seq analysis compared the OCM-treated M1 MФs versus non-OCM-treated M1 MФs. Functional enrichment and pathway analysis of significantly differentially expressed mRNAs. The results show the enriched related pathways. (B and C) Pull-down assays for the active RhoA in OCM-MФs, AS-MФs (PBMC MΦs (upper) or THP-1 MΦs (lower)). (D) Linoleic acid (LA, 200 µM) and methyl arachidonate (MAA, 200 µM), but not oleic acid (OA, 200 µM), led to a reduction in RhoA activity in M0 MФs (PBMC) after 24 h treatment. (E) Knockdown of RhoA is confirmed by Western blotting. After transfecting M0 MΦs (THP-1 MФs) by RhoA siRNAs (#1 - #3) or scrambled siRNA (SC) for 48 h, cellular proteins were collected, and RhoA, YAP1, TAZ, MST1, and β-actin were detected by immunoblotting. (F) The localization of YAP and TAZ in the cytoplasm and nuclear was measured in MФs cells after 48 h post-transfection of RhoA siRNAs (#1 - #3) or scrambled siRNA (SC). (G) The protein expression of YAP1 and MST1 were measured in M0 MФs (PBMC MФs) treated with the control medium, OCM, or OCM-C. The expression of β-actin was used as the internal control. (H) Immunofluorescence analyzed the expression of F-actin (pink) and YAP1 (green) in M0 MΦs treated with the control medium. Nuclei were counterstained with Hoechst (blue). (I) The results of relative F-actin and YAP1 expression in macrophages are shown in the figures. (n = 3). Scale bar: 5 μm. (J) The localization of YAP1 in the cytoplasm and nuclear was measured in M0 MΦs via western blot after OCM stimulation for 24 h. (K) Linoleic acid (LA) reduced nuclear YAP1 activity in a dose-dependent in MΦs after 24 h treatment. XMU MP1 (5µM) restores nuclear YAP1 in LA-treated MΦs