Extended Data Fig. 6: Nrg4 decreased inflammation and migration in macrophages, and inhibited endothelial NF-κB signal through ErbB4 receptor in vivo.
(A) Representative migration images of macrophage. Scale bar, 100μm. (B) Quantitative analysis of (A). (C) The expression levels of NF-κB signaling in MAECs in vitro. (D) Quantitative analysis of (C). (E) The levels of TNF-α, IL-1β and IL-6 in the supernatant, and the mRNA levels in lysate of macrophages. (F) Schematic of the transgenic construct used to generate endothelial-specific ErbB4 knockout animals. (G) The experiment schedule for the effects of the overexpression of Nrg4 in BAT in situ on the protection of endothelial injury in KO and CKO/KO mice (6 mice in each group). (H) MAECs were derived from CKO and WT mice. ErbB4 and VEGFR2 (vascular endothelial growth factor receptor 2) protein expression in MAECs and unselected cell lysates (n = 3). (I) ErbB4 expression in EC, BAT and liver from CKO and WT mice (n = 3). (J) The expression levels of MAECs NF-κB signaling in WT and KO mice with or without overexpression of Nrg4 (n = 6). (K) Quantitative analysis of (J). (L) The expression levels of MAECs NF-κB signaling in WT and CKO/KO mice with or without overexpression of Nrg4 (n = 6). (M) Quantitative analysis of (L). For the macrophages transwell migration assay, the RAW264.7 cells were treated by ox-LDL 100ug/ml for 12 h under the condition with or without rNrg4 100 ng/ml for 48 h. The overexpression of Nrg4 in BAT in situ was performed in KO or CKO/KO mice aged 6 weeks and then fed a WD for 12 weeks (6 mice in each group). For b, k, and m, P values were calculated by two-sided t-test. For d and e, P values were calculated by one-way ANOVA with Tukey’s multiple-comparison test. Data were shown as mean ± SEM. Each experiment was repeated 5 times.*P < 0.001.
