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. Author manuscript; available in PMC: 2022 Jun 29.
Published in final edited form as: Nat Metab. 2022 Jun 27;4(6):739–758. doi: 10.1038/s42255-022-00593-x

Figure 6. pVHL restores cellular oxygen consumption rate.

Figure 6

a, Mitochondrial respiration reflected by oxygen consumption rate (OCR) of 786-O cells with indicated genotype 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-c, The measurement of oxygen consumption rate (OCR) of 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). a-d, data are presented as mean values ± SD. n = 3 biological independent experiments. e, Crystal violet staining of 786-O cells with indicated VHL status treated with high glucose (25 mM) or no glucose (0 mM) respectively for 36 hours. Corresponding ADP/ATP ratio is shown in (h). f, Crystal violet staining of 786-O cells with indicated VHL status treated with 100 μM 3-bromopyruvic acid (3-BP) for 4 hours. Corresponding ADP/ATP ratio is shown in (i). g, Crystal violet staining of 786-O cells with indicated VHL status treated with 25 μM gossypol for 36 hours. Corresponding ADP/ATP ratio is shown in (j). h-j, data are presented as mean values ± SD. 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 indicated VHL status treated with 5 μM PKA inhibitor H89 for 24 hours, prior to glucose deprivation for 36 hours. 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 hours, prior to 100 μM 3-BP treatment for 4 hours. 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 hours, prior to 25 μM gossypol for 36 hours. Corresponding ADP/ATP ratio is shown in (p). n-p, data are presented as mean values ± SD. One way ANOVA Tukey's Multiple Comparison Test. *p <0.05, ****p <0.0001. n = 3 biological independent experiments.