Figure 3
Macrophages promote orbital fibroblasts contractility independently from αSMA levels. (a) Orbital fibroblasts (1.48 × 105 cells/ml) were cultured with macrophages (2.22 × 105 cells/ml) in serum-free medium for 7 days. Gel contraction is shown as mean ± SEM. Control (COs) and GO (HOs) fibroblasts are each averaged from 3 lines (COs: CO4, CO6, CO7; HOs: HO1, HO2, HO4) with n = 3 and triplicates in each experiment. *p < 0.05, **p < 0.01, Mann-Whitney test. (b) Representative western blots and quantification of relative αSMA protein levels for the same 3 COs and 3 HOs cell lines after 5~7 days culture in the presence of 10% serum in monolayers (2D) and in gels (3D), and in serum-free medium in gels (SF) (n ≥ 3; **p < 0.01, Kruskal-Wallis test and post-hoc). Full-length blots are presented in Supplementary Fig. S1. (c) Representative western blot and quantification of αSMA levels showed effective αSMA knockdown by siRNA in HO2 (GO) orbital fibroblasts (n = 4). Full-length blots are presented in Supplementary Fig. S2. (d) Downregulating αSMA significantly decreased the contractility of HO2 fibroblasts in the presence of 10% serum (n = 3 and triplicates in each experiment, Day 7 p < 0.001, Mann-Whitney test). (e) Downregulating αSMA did not decrease macrophage-stimulated contraction of HO2 orbital fibroblasts in serum-free medium (n = 4 and triplicates in each experiment, Day 7 p = 0.215, Mann-Whitney test).
