Figure S3.
ATP levels changes by complex I or III inhibition. (A) Graph of P values for comparisons of GO-ATeam1 timecourses in Fig. 3 A (CCCP or CCCP/CK666 treatment of MEFs in 2 mM glucose). Statistical significance was calculated by two-way ANOVA using Tukey’s multiple comparisons test. (B) ATP levels (± SD) in MEFs upon DMSO, 1 µM CCCP or 1 µM CCCP + 100 µM CK666 treatments in medium containing either 1 or 25 mM glucose without serum, assayed from cell extracts. Points indicate individual measurements starting with 106 cells/dish. n = 12 individual well measurements from four independent experiments for 25 mM glucose groups and 18 individual well measurements from six independent experiments for 1 mM glucose groups. n.s. P > 0.05; **** P < 0.0001. Statistical significance was calculated by one-way ANOVA using Tukey’s multiple comparisons test. (C) Graph of P values for comparisons of GO-ATeam1 timecourses in Fig. 3 B (antimycin A and rotenone treatments of MEFs in 2 mM glucose). Statistical significance was calculated by two-way ANOVA using Tukey’s multiple comparisons test. (D) Graph of change in ATP levels (± SEM) in live MEFs stimulated with 20 µM CCCP in the absence or presence of 100 µM CK666, using GO-ATeam1 biosensor. Cells cultured in medium containing 25 mM glucose with serum. n ≥ 20 cells for each group combined from two independent experiments. Arrow indicates time of treatment. Statistical significance was calculated by two-way ANOVA using Tukey’s multiple comparisons test and presented in Table S1. (E) Graph of change in ATP levels (± SEM) in live MEFs stimulated with 25 µM antimycin A or 50 µM rotenone in the absence or presence of 100 µM CK666, using GO-ATeam1 biosensor. Cells cultured in medium containing 25 mM glucose with serum. n ≥ 33 cells for each group combined from two independent experiments. Arrow indicates time of treatment. Statistical significance was calculated by two-way ANOVA using Tukey’s multiple comparisons test and presented in Table S1. Number of experiments, statistical tests, and sample sizes are provided in Table S1.

ATP levels changes by complex I or III inhibition. (A) Graph of P values for comparisons of GO-ATeam1 timecourses in Fig. 3 A (CCCP or CCCP/CK666 treatment of MEFs in 2 mM glucose). Statistical significance was calculated by two-way ANOVA using Tukey’s multiple comparisons test. (B) ATP levels (± SD) in MEFs upon DMSO, 1 µM CCCP or 1 µM CCCP + 100 µM CK666 treatments in medium containing either 1 or 25 mM glucose without serum, assayed from cell extracts. Points indicate individual measurements starting with 106 cells/dish. n = 12 individual well measurements from four independent experiments for 25 mM glucose groups and 18 individual well measurements from six independent experiments for 1 mM glucose groups. n.s. P > 0.05; **** P < 0.0001. Statistical significance was calculated by one-way ANOVA using Tukey’s multiple comparisons test. (C) Graph of P values for comparisons of GO-ATeam1 timecourses in Fig. 3 B (antimycin A and rotenone treatments of MEFs in 2 mM glucose). Statistical significance was calculated by two-way ANOVA using Tukey’s multiple comparisons test. (D) Graph of change in ATP levels (± SEM) in live MEFs stimulated with 20 µM CCCP in the absence or presence of 100 µM CK666, using GO-ATeam1 biosensor. Cells cultured in medium containing 25 mM glucose with serum. n ≥ 20 cells for each group combined from two independent experiments. Arrow indicates time of treatment. Statistical significance was calculated by two-way ANOVA using Tukey’s multiple comparisons test and presented in Table S1. (E) Graph of change in ATP levels (± SEM) in live MEFs stimulated with 25 µM antimycin A or 50 µM rotenone in the absence or presence of 100 µM CK666, using GO-ATeam1 biosensor. Cells cultured in medium containing 25 mM glucose with serum. n ≥ 33 cells for each group combined from two independent experiments. Arrow indicates time of treatment. Statistical significance was calculated by two-way ANOVA using Tukey’s multiple comparisons test and presented in Table S1. Number of experiments, statistical tests, and sample sizes are provided in Table S1.

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