Extended Data Figure 4. Downstream metabolic effects of fructose metabolism in NMCs.

a, log₂ fold change of metabolites in NMCs overexpressing KHK-C and cultured with glucose or glucose and fructose compared to the corresponding control transduced NMCs (n = 4 biological replicates per group). b, Incorporation of [³H]fructose into RNA (top), DNA (middle) and protein (bottom) of NMCs treated as indicated (n = 4 biological replicates per group). c, Uptake of [¹⁴C]deoxyglucose in NMCs infected as in b at depicted time points (n = 4 biological replicates per group). d, [³H]leucine incorporation in NMCs transduced with shNs or shKhk and co-transduced with either empty overexpression vector or KHK-A or KHK-C respectively. NMCs were cultured in media with increasing fructose concentrations, under physiologic glucose amounts or with increasing glucose concentrations under physiologic fructose amounts. Data are presented relative to shNs/empty-vector-transduced NMCs at 5 mM glucose/0 μM fructose (n = 4 biological replicates). e, ADP/ATP ratios in NMCs transduced as indicated. (n = 4 biological replicates, data show 1 of 3 representative experiments). f, Immunofluorescence images of NMCs transduced with empty vector (upper panel) or HIF1αΔODD (middle panel) lentiviruses or exposed to hypoxia (lower panel) were additionally transduced as indicated. Prior to staining for sarcomeric α-actinin, Oil Red O and DAPI, NMCs were incubated with oleic acid. g, Quantification of lipid droplets/cell in NMCs of immunofluorescent images shown in f. h, Ratio of lipid droplets/cell area of NMCs shown in g. i–l, NMCs were infected as indicated and processed for oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements under basal conditions or after injection of OA or FCCP. Depicted are rates expressed as OCR to ECAR ratios (upper panels) or individual ECAR (for measurements at baseline) or OCR values (n = 8 biological replicates for HIF1αΔODD/shKhk-A; n = 10 biological replicates for all other groups). Error bars are s.d. (b–e) or s.e.m. (g–l). *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA followed by Dunnett’s multiple comparison post-test (b–e, g, h). Two-tailed unpaired t-test (i–l).