Fig. 6: pVHL restores cellular oxygen consumption rate.
a, Mitochondrial respiration reflected by OCR of 786-O cells with the indicated genotypes was monitored using the Seahorse XF-96 Extracellular Flux Analyzer with the sequential injection of oligomycin (1 μM), FCCP (1 μM) and rotenone/antimycin (0.5 μM). b–d, OCR measurement in 786-O cells with indicated VHL status transduced with lentiviral pL.KO shRNA targeting EGLN3 or no targeting control (b), primary EGLN3+/+ and EGLN3−/− MEFs (c) stably transduced with lentivirus encoding EglN3 WT, catalytic death mutant or empty control (d). In a–d, data are presented as mean values ± s.d. n = 3 biological independent experiments. e, Crystal violet staining of 786-O cells with the indicated VHL status treated with high glucose (25 mM) or no glucose (0 mM) for 36 h. Corresponding ADP/ATP ratio is shown in h. f, Crystal violet staining of 786-O cells with the indicated VHL status treated with 100 μM 3-BP for 4 h. Corresponding ADP/ATP ratio is shown in i. g, Crystal violet staining of 786-O cells with the indicated VHL status treated with 25 μM gossypol for 36 h. Corresponding ADP/ATP ratio is shown in j. In h–j, data are presented as mean values ± s.d. One-way ANOVA Tukey’s multiple-comparison test. *P < 0.05, ****P < 0.0001. n = 3 biological independent experiments. k, Crystal violet staining of 786-O cells with the indicated VHL status treated with 5 μM PKA inhibitor H89 for 24 h, before glucose deprivation for 36 h. Corresponding ADP/ATP ratio is shown in n. l, Crystal violet staining of 786-O cells treated with 5 μM PKA inhibitor H89 for 24 h, before 100 μM 3-BP treatment for 4 h. Corresponding ADP/ATP ratio is shown in o. m, Crystal violet staining of 786-O cells treated with 5 μM PKA inhibitor H89 for 24 h, before 25 μM gossypol for 36 h. Corresponding ADP/ATP ratio is shown in p. In n–p, data are presented as mean values ± s.d. One-way ANOVA Tukey’s multiple-comparison test. *P < 0.05, ****P < 0.0001. n = 3 biological independent experiments.
