Supplementary Figure 2: Inhibition of NAMPT affects glycolytic metabolism of inflammatory macrophages due to diminished limiting of GAPDH activity by NAD⁺.

(a-f) M(γ+LPS) or M(LPS) polarized for 18 h with or without FK866 and subsequently analyzed. (a,b) ECAR after glucose injection (a: n = 5 technical replicates, b: n = 4 technical replicates, both representative of over ten independent experiments). (c,d) Lactate production (n = 3 biologically independent samples, representative of two independent experiments). (e,f) basal OCR (e: n = 4 technical replicates, f: n = 4 technical replicates, both are representative of over ten independent experiments. (g-j) M(γ+LPS) or M(LPS) polarized for 18 h with vehicle control, FK866, GPP78 or STF118804 and subsequently analyzed. Real time changes in the ECAR (g) and ECAR after glucose injection (h) (n = 5 technical replicates, representative of three independent experiments). NAD⁺ as quantified by LC-MS (i) and ATP levels (j) (n = 4 biologically independent samples, representative of two independent experiments). (k, l) Volcano plots showing differentially expressed genes in M(γ+LPS) (k) or M(LPS) (l) untreated cells compared to FK866 treated, highlighting key genes in the glycolytic pathway as obtained from the Kyoto Encyclopedia of genes and genomes (pathway identifier: 00010) (n = 3 biologically independent samples). (m) LC-MS quantitation of glycolytic metabolites in control M(LPS) or M(LPS) + FK866 (n = 4 biologically independent samples, representative of three independent experiments). (n-q) Macrophages were polarized for 18h with or without 1 μM heptelidic acid (HA). (n) Basal ECAR, and basal OCR of M(γ+LPS) (n = 4 technical replicates, representative of two independent experiments). (o) ATP quantification of M(γ+LPS) (n = 3 biologically independent samples, representative of two independent experiments). (p) Basal ECAR, and basal OCR of M(LPS) (n = 4 technical replicates, representative of two independent experiments). (q) ATP quantification of M(LPS) (n = 3 biologically independent samples, representative of two independent experiments). (r) Western Blot showing NAMPT and β-actin expression in macrophages transduced with empty vector (EV) shRNA or Nampt shRNA. The blot was cropped to show relevant bands, and is representative of four independent experiments with similar results. (s) Viability of macrophages transduced with EV shRNA or Nampt shRNA, polarized for 18 h with γ+LPS and assessed by flow cytometry (n = 3 biologically independent samples, representative of three independent experiments). Error bars are mean ± SEM. Data were analyzed in c,d,m,o,q,s by unpaired, two-sided Student’s t-test and in h-j by one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, ***p < 0.001 and ****p < 000.1