Extended Data Fig. 3: Mannose attenuates LIF release but does not substitute for glucose in restoring all metabolite pools.
A. Cell death measured by PI-positivity in A549 cells after incubation for 24 h at different glucose concentrations (n = 3). B. Protein content per well in A549 cells after 24 h incubation in glucose deprived conditions with re-addition of several glucose derived metabolites (n = 6, 4 for Man-ol condition). Glc=glucose; Man= mannose; Fru=fructose; Gal=galactose; GlcNAc= N-acetyl-glucosamine; Lac=lactate; Pyr=pyruvate, Man-ol=mannitol. C. LIF protein secretion (top) and protein content per well (bottom) in H1299 cells measured using ELISA after 24 h incubation in glucose deprived conditions with re-addition of several glucose derived metabolites (n = 3, 4 for Glc, Man and Man-ol condition). Glc=glucose; Man= mannose; Fru=fructose; Gal=galactose; GlcNAc= N-acetyl-glucosamine; Lac=lactate; Pyr=pyruvate, Man-ol=mannitol. D. LIF protein secretion (top) and protein content per well (bottom) in SW900 cells measured using ELISA after 24 h incubation in glucose deprived conditions with re-addition of several glucose derived metabolites (n = 3, and 5 for Glc and GlcNAc conditions). Glc=glucose; Man= mannose; Fru=fructose; Gal=galactose; GlcNAc= N-acetyl-glucosamine; Lac=lactate; Pyr=pyruvate, Man-ol=mannitol. E. Diagram depicting the fate of labelled mannose when glucose is present (left) or absent (right). Font size reduction indicated reduced pools, color in arrows and fonts indicate labelling. F. Total content of the indicated metabolites in A549 cells from the experiment shown in Fig. 3 (3 h - GC-MS, n = 3). G. LIF secreted from H1299 or SW900 cells after 24 h treatment with the NADPH replenisher N-acetylcysteine (NAC) measured using ELISA. Results normalized by protein content (n = 3). H. LIF secreted from H1299 cells after 24 h treatment with the inhibitor of glycolysis (3PO), measured using ELISA (n = 3). I. Representative WB of A549 cells at the times and conditions indicated. β-actin (“β-Act”) as loading control (n = 2). J. Protein content per well measured by BCA of panel Fig. 3i (n = 3). K. LIF secreted by H1299 cells after 24 h treatment with activator of AMPK, A769662 (A76) measured using ELISA. Results normalized by protein content (n = 2). L. Representative WB of A549 cells at 24 h and indicated conditions. Ponceau was used as loading control (n = 2). Graphs show average values and individual replicates, and error bars represent the standard error of the mean (SEM); statistical significance was determined by one-way ANOVA with multiple comparisons test except for panel F, which was analyzed by one-way ANOVA plus Tukey’s Test for Post-hoc Analysis.
