Figure S11.
Effect of CK666 on ADA and glycolytic activation in Teff. (A) Teff stained for actin filaments (TRITC-phalloidin, green), mitochondria (Tom20, red) and DNA (DAPI, blue) after CCCP, antimycin A, rotenone, or hypoxia in the presence of CK666 for 2 mM glucose medium without serum (1 µM CCCP + 100 µM CK666, 3 min; 2.5 µM antimycin A + 100 µM CK666 and 5 µM rotenone + 100 µM CK666, 5 min; hypoxia with 100 µM CK666, 60 min). Images at right are zooms of boxed regions. Scale bars: 5 μm (full cell) and 2 μm (inset). (B) ECAR (± SD) in 25 mM glucose without serum upon 100 µM CK666, 1 µM CCCP or 1 µM CCCP + 100 µM CK666 addition at 45 min, then 50 mM 2-deoxyglucose (2-DG) at 223 min. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (C) ECAR (± SD) in 25 mM glucose without serum upon DMSO, 2.5 µM antimycin A or 2.5 µM antimycin A + 100 µM CK666 addition at 45 min, then 50 mM 2-DG at 223 min. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (D) ECAR (± SD) in 25 mM glucose without serum upon DMSO, 5 µM rotenone or 5 µM rotenone + 100 µM CK666 addition at 45 min, then 50 mM 2-DG at 223 min. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment, and blue arrow indicates 2-DG treatment. (E) OCR (± SD) in 2 mM glucose without serum upon 100 µM CK666, 1 µM CCCP or 1 µM CCCP + 100 µM CK666 addition at 45 min, then 50 mM 2-DG at 223 min. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (F) OCR (± SD) in 2 mM glucose without serum upon DMSO, 2.5 µM antimycin A or 2.5 µM antimycin A + 100 µM CK666 addition at 45 min, then 50 mM 2-DG at 223 min. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (G) OCR (± SD) in 2 mM glucose without serum upon DMSO, 5 µM rotenone or 5 µM rotenone + 100 µM CK666 addition at 45 min, then 50 mM 2-DG at 223 min. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. Number of experiments and sample sizes are provided in Table S1.

Effect of CK666 on ADA and glycolytic activation in T eff . (A) Teff stained for actin filaments (TRITC-phalloidin, green), mitochondria (Tom20, red) and DNA (DAPI, blue) after CCCP, antimycin A, rotenone, or hypoxia in the presence of CK666 for 2 mM glucose medium without serum (1 µM CCCP + 100 µM CK666, 3 min; 2.5 µM antimycin A + 100 µM CK666 and 5 µM rotenone + 100 µM CK666, 5 min; hypoxia with 100 µM CK666, 60 min). Images at right are zooms of boxed regions. Scale bars: 5 μm (full cell) and 2 μm (inset). (B) ECAR (± SD) in 25 mM glucose without serum upon 100 µM CK666, 1 µM CCCP or 1 µM CCCP + 100 µM CK666 addition at 45 min, then 50 mM 2-deoxyglucose (2-DG) at 223 min. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (C) ECAR (± SD) in 25 mM glucose without serum upon DMSO, 2.5 µM antimycin A or 2.5 µM antimycin A + 100 µM CK666 addition at 45 min, then 50 mM 2-DG at 223 min. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (D) ECAR (± SD) in 25 mM glucose without serum upon DMSO, 5 µM rotenone or 5 µM rotenone + 100 µM CK666 addition at 45 min, then 50 mM 2-DG at 223 min. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment, and blue arrow indicates 2-DG treatment. (E) OCR (± SD) in 2 mM glucose without serum upon 100 µM CK666, 1 µM CCCP or 1 µM CCCP + 100 µM CK666 addition at 45 min, then 50 mM 2-DG at 223 min. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (F) OCR (± SD) in 2 mM glucose without serum upon DMSO, 2.5 µM antimycin A or 2.5 µM antimycin A + 100 µM CK666 addition at 45 min, then 50 mM 2-DG at 223 min. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (G) OCR (± SD) in 2 mM glucose without serum upon DMSO, 5 µM rotenone or 5 µM rotenone + 100 µM CK666 addition at 45 min, then 50 mM 2-DG at 223 min. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. Number of experiments and sample sizes are provided in Table S1.

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