Figure 3.
ADA is required for glycolytic activation upon mitochondrial perturbation in MEFs. (A) Cytoplasmic ATP levels (± SEM) after 20 µM CCCP in the absence or presence of 100 µM CK666, using GO-ATeam1. n ≥ 35 cells per group combined from two independent experiments. P values are graphed in Fig. S3 A. Arrow indicates time of treatment. Experiments done in 2 mM glucose with serum. (B) Cytoplasmic ATP levels (± SEM) after 25 µM antimycin A or 50 µM rotenone in the absence or presence of 100 µM CK666, using GO-ATeam1. n ≥ 24 cells per group combined from two independent experiments. P values graphed in Fig. S3 C. Arrow indicates time of treatment. Experiments done in 2 mM glucose with serum. (C) ECAR (± SD) upon 100 µM CK666, 1 µM CCCP or 1 µM CCCP + 100 µM CK666 addition (15 min), followed by 50 mM 2-deoxyglucose (2-DG) (59 min) in 2 mM glucose medium without serum. n = 3 individual well measurements for CCCP and CK666; 4 for CCCP + CK666. Pink arrow indicates drug treatment and blue arrow indicate 2-DG treatment. (D) ECAR (± SD) upon DMSO, 100 µM CK666, 2.5 µM antimycin A or 2.5 µM antimycin A + 100 µM CK666 addition (33 min), then 50 mM 2-DG (258 min) in 2 mM glucose medium without serum. n = 5 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (E) ECAR (± SD) upon DMSO, 100 µM CK666, 5 µM rotenone or 5 µM rotenone + 100 µM CK666 addition (33 min), then 50 mM 2-DG (258 min) in 2 mM glucose medium without serum. n = 5 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (F) Effect of glucose concentration on ECAR spike (± SD) induced by 3 min 1 µM CCCP, with and without 100 µM CK666. n = 9 individual well measurements for CCCP and CK666; 12 for CCCP + CK666. P values graphed in Fig. S4 D. (G) Effect of 100 µM CK666 on lactate production in hypoxia (1% O2) in MEFs at 2 mM glucose without serum. Points indicate individual well measurements starting with 100,000 cells/well. n = 8 individual well measurements from four independent experiments. ** P = 0.0018; **** P < 0.0001. Statistical significance was calculated by two-way ANOVA using Tukey’s multiple comparisons test. Number of experiments, FOV, sample sizes and statistical tests are provided in Table S1.

ADA is required for glycolytic activation upon mitochondrial perturbation in MEFs. (A) Cytoplasmic ATP levels (± SEM) after 20 µM CCCP in the absence or presence of 100 µM CK666, using GO-ATeam1. n ≥ 35 cells per group combined from two independent experiments. P values are graphed in Fig. S3 A. Arrow indicates time of treatment. Experiments done in 2 mM glucose with serum. (B) Cytoplasmic ATP levels (± SEM) after 25 µM antimycin A or 50 µM rotenone in the absence or presence of 100 µM CK666, using GO-ATeam1. n ≥ 24 cells per group combined from two independent experiments. P values graphed in Fig. S3 C. Arrow indicates time of treatment. Experiments done in 2 mM glucose with serum. (C) ECAR (± SD) upon 100 µM CK666, 1 µM CCCP or 1 µM CCCP + 100 µM CK666 addition (15 min), followed by 50 mM 2-deoxyglucose (2-DG) (59 min) in 2 mM glucose medium without serum. n = 3 individual well measurements for CCCP and CK666; 4 for CCCP + CK666. Pink arrow indicates drug treatment and blue arrow indicate 2-DG treatment. (D) ECAR (± SD) upon DMSO, 100 µM CK666, 2.5 µM antimycin A or 2.5 µM antimycin A + 100 µM CK666 addition (33 min), then 50 mM 2-DG (258 min) in 2 mM glucose medium without serum. n = 5 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (E) ECAR (± SD) upon DMSO, 100 µM CK666, 5 µM rotenone or 5 µM rotenone + 100 µM CK666 addition (33 min), then 50 mM 2-DG (258 min) in 2 mM glucose medium without serum. n = 5 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (F) Effect of glucose concentration on ECAR spike (± SD) induced by 3 min 1 µM CCCP, with and without 100 µM CK666. n = 9 individual well measurements for CCCP and CK666; 12 for CCCP + CK666. P values graphed in Fig. S4 D. (G) Effect of 100 µM CK666 on lactate production in hypoxia (1% O2) in MEFs at 2 mM glucose without serum. Points indicate individual well measurements starting with 100,000 cells/well. n = 8 individual well measurements from four independent experiments. ** P = 0.0018; **** P < 0.0001. Statistical significance was calculated by two-way ANOVA using Tukey’s multiple comparisons test. Number of experiments, FOV, sample sizes and statistical tests are provided in Table S1.

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