Supplementary Figure 3: Modulation of inflammatory macrophage phenotype by an NAMPT inhibitor is linked to changes in glycolytic metabolism.

(a) Example flow cytometric gating strategy used to identify live, CD11b⁺, F4/80⁺ macrophages. (b) Intracellular staining for TNF production in M0, M(γ+LPS) and M(γ+LPS) + FK866 as assessed by flow cytometry after 8 h of polarization, representative flow cytomtery plots with indicated percentage of TNF⁺ cells and SEM (n = 3 biologically independent samples, representative of two independent experiments). (c) Heatmap of antigen presentation and pro-inflammatory gene expression in macrophages polarized for 18 h as indicated. Statistically significant (adjusted p value < 0.1) up- or down-regulated (>2 fold) genes denoted by * in M(γ+LPS) compared to M(γ+LPS) + FK (n = 3 biologically independent samples). (d,e) M(γ+LPS) polarized with vehicle control or 50 nM FK866, GPP78 or STF118804 for 18h and assessed by flow cytometry. (d) Viability relative to control treated M(γ+LPS) (n = 4 biologically independent samples, representative of three independent experiments). (e) CD80, NOS2, and MHC class II expression (n = 3 biologically independent samples, representative of three independent experiments). (f) Expression of NOS2 and production of IL-6 and TNFα of M(γ+LPS) polarized for 18h with or without 1 μM HA and measured by flow cytometry and ELISA, respectively (n = 3 biologically independent samples, representative of two independent experiments). (g) Expression of markers RELMα, CD206 and CD301 in M(IL-4) polarized for 18h with or without FK866 treatment, analyzed by flow cytometry (n = 3 biologically independent samples, representative of three independent experiments). Error bars are mean ± SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 000.1.