Supplementary Figure 1: Inflammatory macrophages are depleted of NAD⁺, and NAD⁺ salvage is required for viability.

(a) NAD⁺ in M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 18 h, quantified by NAD cycling assay (n = 3 biologically independent samples, representative of three independent experiments). (b) NADH, NADP⁺, NADPH in M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 18 h, as quantified by LC-MS (n = 3 biologically independent samples, representative of three independent experiments). (c) Expression of NAMPT mRNA in macrophages polarized as indicated for 18h, normalized to mRNA encoding HPRT and presented relative to M0 control cells, set as 1 (n = 3 biologically independent samples, representative of three independent experiments). (d) RNA-seq analysis of expressed enzymes in the Preiss Handler Pathway or de novo NAD⁺ synthesis pathway in M0, M(γ+LPS), M(LPS) and M(IL-4) polarized for 18 h. Statistically significant (adjusted p value < 0.1) upregulated (> 2 fold) genes denoted by * in M(γ+LPS) polarized cells compared to M0 (n = 3 biologically independent samples). (e) NADH levels of M0, M(γ+LPS), M(LPS) and M(IL-4) cultured for 18 h in the presence or absence of 50 nM FK866, relative to M0 (n = 3 biologically independent samples, representative of two independent experiments). (f) Viability of M(γ+LPS) treated with increasing FK866 concentrations from 20 nM – 1600 nM as determined by flow cytometry, presented as percentage of vehicle treated control (n = 3 biologically independent samples, representative of three independent experiments). (g) NAD⁺ of bone marrow macrophages derived from C57Bl/6N mice. M0, M(γ+LPS) +/- 50 nM FK866, M(LPS) +/- 50nM FK866 and M(IL-4) cultured for 18 h, and NAD⁺ quantified by LC-MS (n = 3 biologically independent samples, representative of two independent experiments). Error bars are mean ± SEM. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, ***p < 0.001 and ****p < 000.1.