Supplementary Figure 8: Inhibition of complex III and ROS scavenging affect inflammatory macrophage function.

(a) Viability of M(γ+LPS) polarized in the presence of CI inhibitor (Rotenone), CIII Qi inhibitor (Antimycin A) or a Complex III Qo inhibitor (Myx) for 1 h then harvested and assessed by flow cytometry. Viability presented as percentage control M(γ+LPS) (n = 3 biologically independent samples, representative of five independent experiments). (b) NAD⁺ and NADH quantification by LCMS in M(γ+LPS) polarized for 1 h with or without Myx (n = 3 biologically independent samples, representative of three independent experiments). (c) Viability of M(γ+LPS) at 18 h, after treatment with Myx or vehicle control for the first hour of polarization. Viability shown relative to vehicle control M(γ+LPS) (n = 3 biologically independent samples, representative of three independent experiments). (d,e) M(γ+LPS) were polarized with or without 10 mM NAC for 18 h then analyzed. (d) CD80, NOS2, MHC class II expression (n = 4 biologically independent samples, representative of three independent experiments). (e) IL-6 and TNF production (n = 3 biologically independent samples, representative of three independent experiments). (f,g) Macrophages were pre-treated for 2 h with NAC then stimulated with γ+LPS, 1 h later cells were washed and fresh γ+LPS added. 18 h later cells were analysed. (f) CD80, NOS2, MHC class II expression (n = 4 biologically independent samples, representative of two independent experiments). (g) IL-6 and TNF production (n = 4 biologically independent samples, representative of two independent experiments). Error bars are mean ± SEM. Data were analyzed by unpaired, two-tailed students t-test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 000.1.