Fig. 5: NAMPT enzymatic function is required for LPS/IFNγ-induced glycolysis at GAPDH.

a Glycolytic stress test showing secreted lactate as measured by ECAR (extracellular acidification rate) for BMMs treated with combinations of LPS/IFNγ and FK866. Error bars represent SEM for N = 10 biological replicates. b Glycolytic and TCA cycle intermediate metabolites in BMMs treated with combinations of LPS/IFNγ and FK866. Values on Y-axis are Log₂ ratios of LPS/IFNγ induced metabolite levels normalized as Fold Change vs. non-inducing medium only (black), or medium with FK866 only (red). Blue background indicates those steps prior to the first NAD requiring GAPDH step, and red background indicates those steps after GAPDH. X-axis categories are metabolites in pathway order as in Supplementary Fig. 5A⁸⁶. Statistical significance for the effects of FK866 on induction are reported in Supplementary Fig. 5B. c Expression of individual glycolytic gene set members as measured by RT-qPCR from the indicated macrophage RNAs when treated with combinations of LPS/IFNγ and FK866, showing reduction of inducible expression in the presence of FK866-inhibited NAMPT. d Expression of the same glycolytic gene set members as in c from Vav-Cre alone or Vav-Cre NRE1 fl/fl (NRE1-KO) macrophage RNAs treated with LPS/IFNγ, showing loss of induction in the absence of the NRE1. e Inducible glycolytic and f inflammatory gene expression is lost in the presence of the glycolysis inhibitor 2-Deoxy-D-Glucose. Results presented are the average of N = 3 biological replicates. Except for a, b, e, f, data are representative of at least three independent experiments. For b–f, results presented are the average of N = 3 biological replicates. p values were determined by a two-tailed unpaired t-test: *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.00005. Error bars represent SEM.