Extended Data Fig. 6. VHL decreases glycolysis.

(a) Extracellular acidification rate (ECAR) of 786-O cells with indicated genotype was monitored using the Seahorse XF-96 Extracellular Flux Analyzer with the sequential injection of glucose (10 mM), oligomycin (1 μM) and 2-deoxy-glucose (2-DG) (50 μM). The rates of glycolysis and glycolysis capacity were respectively quantified by normalization of amount of cells. One way ANOVA Tukey's Multiple Comparison Test. **p =0.003, ***p =0.0002, ****p <0.0001. (b) Extracellular acidification rate (ECAR) of 786-O cells with indicated VHL status transduced with lentiviral pL.KO shRNA targeting EGLN3 or no targeting control was measured as described above. The rates of glycolysis and glycolysis capacity were respectively quantified by normalization of amount of cells. One way ANOVA Tukey's Multiple Comparison Test. ****p <0.0001. (c) Extracellular acidification rate (ECAR) of primary EGLN3+/+ and EGLN3-/- MEFs. The rates of glycolysis and glycolysis capacity were respectively quantified by normalization of amount of cells. One way ANOVA Tukey's Multiple Comparison Test. ****p <0.0001. (d) Extracellular acidification rate (ECAR) of primary EGLN3-MEFs of indicated genotype stably transduced with lentivirus encoding EGLN3 WT, catalytic death mutant or empty control was monitored as described above. The rates of glycolysis and glycolysis capacity were respectively quantified by normalization of amount of cells. One way ANOVA Tukey's Multiple Comparison Test. ****p <0.0001. a-d, data are presented as mean values ± SD. n = 3 biological independent experiments.