Extended Data Fig. 8: Nrg4 inhibited inflammation and adhesion response in MAECs via ErbB4/Akt/NF-κB signal in vitro.
(A) The levels of IκBα, P-IκBα, Akt, p-Akt, p65 and P-p65 expressions in the MAECs under different conditions. (B) Quantitative analysis of P-Akt, P-IκBα and P-p65 in (A). GAPDH was used as a loading control. (C) The levels of VCAM-1, ICAM-1, E-selectin, IL-1β, IL-6 and TNF-α expressions in the MAECs under different conditions. (D) Cells were seeded onto transwell chambers. Confluent cells were treated with rNrg4 or ox-LDL, and FITC-labelled dextran that migrated through the MAECs monolayer was measured. The data represent the mean fluorescence intensity±SEM. (E) THP-1 monocytes were stained with calcein green and adhered to ox-LDL activated MAECs for 30 min. Fluorescence microscopy was used to measure the number of adherent THP-1 monocytes per microscopic field (x100). (F) Representative images of apoptotic cells in MAECs. (G) Quantitative analysis of (F). (H) The vasodilation responses of human arterial rings to Ach (n = 3). (I, J) NF-κB -luciferase and SV40-Renilla were transfected into MAECs after treatment with si-Control, si-ErbB4 (I), or si-Akt-scramble, si-Akt (J). Cells were left untreated or treated with rNrg4, or/ and ox-LDL before luciferase and Renilla assessment. (K-M) MAECs from WT mice were transfected with si-control and si-ErbB4, then treated with or without rNrg4, or/ and ox-LDL, and IgG, p65 and histone antibodies were used for ChIP assay, and RT–PCR was performed to amplify (K) VCAM-1, (L) E-selectin and (M) IkBa promoters. (N-P) MAECs from WT mice were transfected with si-Akt-scramble and si-Akt, then treated with or without rNrg4, or/ and ox-LDL, and IgG, p65 and histone antibodies were used to ChIP and RT–PCR was performed to amplify (N) VCAM-1, (O) E-selectin and (P) IkBa promoters. MAECs from WT mice were pre-treated with si-RNA, then rNrg4 for 48 h, ox-LDL for 12 h or SC79 for 6 h. For b, c, g and h, P values were calculated by one-way ANOVA with Tukey’s multiple-comparison test. For d, e, i-p, P values were calculated by two-sided t-test or one-way ANOVA with Tukey’s multiple-comparison test. Each experiment was repeated 5 times. *P < 0.01, **P < 0.001, #P < 0.05 vs. Ox-LDL group, ##P < 0.01 vs. Ox-LDL group, ###P < 0.001 vs. Ox-LDL group, †P < 0.05 vs. Nrg4 group, ††P < 0.01 vs. Nrg4 group, ††† P < 0.001 vs. Nrg4 group.
