Figure 2.
ADA stimulation by mitochondrial depolarization or ETC inhibition. (A) Mitochondrial depolarization (assessed by TMRE fluorescence) in MEFs with DMSO, CCCP, antimycin A or rotenone (± SEM) treatments. n ≥ 98 cells per group combined from three independent experiments. Experiments done in 25 mM glucose with serum. (B) MEFs stained for actin filaments (TRITC-phalloidin, green), mitochondria (Tom20, red) and DNA (DAPI, blue) after 3 min treatment with DMSO, 20 µM CCCP, 25 µM antimycin A or 50 µM rotenone in the absence (top) or presence (bottom) of 100 µM CK666. Right images are zooms of boxed regions. Scale bar: 5 μm. (C) % cells (± SD) displaying ADA for the conditions shown in B. n ≥ 62/18 cells/fields of view (FOV) per group combined from two experiments. Experiments done in 25 mM glucose with serum. (D) MEFs stained similarly to B, in normoxia or hypoxia for 30 min, in the presence or absence of 100 µM CK666. Scale bars are 10 μm (full cell) and 5 μm (inset). (E) % cells displaying ADA after 30 min normoxia or hypoxia, in the absence or presence of 100 µM CK666. n ≥ 174/20 cells/FOV per group combined from two biological experiments. Experiments done in 25 mM glucose without serum. Exact number of experiments, FOV and sample size are provided in Table S1.

ADA stimulation by mitochondrial depolarization or ETC inhibition. (A) Mitochondrial depolarization (assessed by TMRE fluorescence) in MEFs with DMSO, CCCP, antimycin A or rotenone (± SEM) treatments. n ≥ 98 cells per group combined from three independent experiments. Experiments done in 25 mM glucose with serum. (B) MEFs stained for actin filaments (TRITC-phalloidin, green), mitochondria (Tom20, red) and DNA (DAPI, blue) after 3 min treatment with DMSO, 20 µM CCCP, 25 µM antimycin A or 50 µM rotenone in the absence (top) or presence (bottom) of 100 µM CK666. Right images are zooms of boxed regions. Scale bar: 5 μm. (C) % cells (± SD) displaying ADA for the conditions shown in B. n ≥ 62/18 cells/fields of view (FOV) per group combined from two experiments. Experiments done in 25 mM glucose with serum. (D) MEFs stained similarly to B, in normoxia or hypoxia for 30 min, in the presence or absence of 100 µM CK666. Scale bars are 10 μm (full cell) and 5 μm (inset). (E) % cells displaying ADA after 30 min normoxia or hypoxia, in the absence or presence of 100 µM CK666. n ≥ 174/20 cells/FOV per group combined from two biological experiments. Experiments done in 25 mM glucose without serum. Exact number of experiments, FOV and sample size are provided in Table S1.

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