Figure 5.
Effector T lymphocytes (Teff) require ADA for glycolytic activation. (A) Teff stained for actin filaments (TRITC-phalloidin, green), mitochondria (Tom20, red), and DNA (DAPI, blue) under un-stimulated conditions, or after treatment with 3 min 1 µM CCCP, 5 min 2.5 µM antimycin A, 5 5 µM min rotenone, or 60 min hypoxia in 2 mM glucose medium. Right images are zooms of boxed regions. Scale bars: 5 μm (full cell) and 2 μm (inset). (B) % cells (± SEM) displaying ADA in treatments described in A, n ≥ 61/5 cells/FOV per group combined from two independent experiments. **** P < 0.0001. Statistical significance between respective treatments in the presence or absence of CK666 was calculated using unpaired two-tailed t tests. Experiments done in 2 mM glucose without serum. (C) ECAR (± SD) in Teff upon addition of 100 µM CK666, 1 µM CCCP or 1 µM CCCP + 100 µM CK666 (45 min), followed by 50 mM 2-deoxyglucose (2-DG; 223 min) in 2 mM glucose medium without serum. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (D) ECAR (± SD) in Teff upon addition of DMSO, 2.5 µM antimycin A or 2.5 µM antimycin A + 100 µM CK666 (45 min), followed by 50 mM 2-DG (223 min) in 2 mM glucose medium without serum. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (E) ECAR (± SD) in Teff upon addition of DMSO, 5 µM rotenone or 5 µM rotenone + 100 µM CK666 (45 min), followed by 50 mM 2-DG (223 min) in 2 mM glucose medium without serum. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (F) Lactate production (2 mM glucose without serum) induced by hypoxia (1% oxygen) in Teff in the presence or absence of 100 µM CK666 addition. Circles indicate individual well measurements starting with 400,000 cells/well. n = 8 individual well measurements from four independent experiments. ** P = 0.0013; **** P < 0.0001. Statistical significance was calculated by two-way ANOVA using Tukey’s multiple comparisons test. Number of experiments, FOV, sample size, and statistical tests used are provided in Table S1.

Effector T lymphocytes (T eff ) require ADA for glycolytic activation. (A) Teff stained for actin filaments (TRITC-phalloidin, green), mitochondria (Tom20, red), and DNA (DAPI, blue) under un-stimulated conditions, or after treatment with 3 min 1 µM CCCP, 5 min 2.5 µM antimycin A, 5 5 µM min rotenone, or 60 min hypoxia in 2 mM glucose medium. Right images are zooms of boxed regions. Scale bars: 5 μm (full cell) and 2 μm (inset). (B) % cells (± SEM) displaying ADA in treatments described in A, n ≥ 61/5 cells/FOV per group combined from two independent experiments. **** P < 0.0001. Statistical significance between respective treatments in the presence or absence of CK666 was calculated using unpaired two-tailed t tests. Experiments done in 2 mM glucose without serum. (C) ECAR (± SD) in Teff upon addition of 100 µM CK666, 1 µM CCCP or 1 µM CCCP + 100 µM CK666 (45 min), followed by 50 mM 2-deoxyglucose (2-DG; 223 min) in 2 mM glucose medium without serum. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (D) ECAR (± SD) in Teff upon addition of DMSO, 2.5 µM antimycin A or 2.5 µM antimycin A + 100 µM CK666 (45 min), followed by 50 mM 2-DG (223 min) in 2 mM glucose medium without serum. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (E) ECAR (± SD) in Teff upon addition of DMSO, 5 µM rotenone or 5 µM rotenone + 100 µM CK666 (45 min), followed by 50 mM 2-DG (223 min) in 2 mM glucose medium without serum. n = 4 individual well measurements per condition. Pink arrow indicates drug treatment and blue arrow indicates 2-DG treatment. (F) Lactate production (2 mM glucose without serum) induced by hypoxia (1% oxygen) in Teff in the presence or absence of 100 µM CK666 addition. Circles indicate individual well measurements starting with 400,000 cells/well. n = 8 individual well measurements from four independent experiments. ** P = 0.0013; **** P < 0.0001. Statistical significance was calculated by two-way ANOVA using Tukey’s multiple comparisons test. Number of experiments, FOV, sample size, and statistical tests used are provided in Table S1.

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