mGluR3, CD63, and cellular oxygen consumption. (a–c) MDA-MB-231 cells were transfected with siRNAs targeting Rab27a/b, CD63, or nontargeting (NT) siRNAs or were left untransfected and incubated in the presence (LY95) or absence (Ctrl) of LY95 (0.3 µM) for 5 d (a and b) or 18 h (c). Oxygen consumption was determined using the Seahorse XFe96 Extracellular Flux Analyzer. Values represent mean ± SEM of three independent experiments. The basal and maximal (Max) OCRs were extracted from data such as those displayed in Fig. 2 a. Maximum OCR was determined by subtracting rotenone/antimycin A (Rot+AntA) OCR from basal OCR values. Values represent the mean of three independent experiments; error bars signify SEM. *, P < 0.033; **, P < 0.01; ***, P < 0.001; paired Student’s t test. Mitochondrial mass was determined using the fluorescent mitochondrial probe MitoTracker Green, and the mean fluorescence intensity per cell was quantified using flow cytometry; values are mean ± SEM of n = 3 (b). TMRE (50 nM) was added to cells for 30 min before analysis by flow cytometry, and CCCP was added for 16 h before this as a positive control for loss of TMRE fluorescence through uncoupling of the mitochondria. Median fluorescence intensities (MFIs) are plotted relative to control; values are SEM of n = 3. (d) Western blotting was used to confirm the effectiveness of siRNAs for CD63 and Rab27 5 d following transfection with actin used as a loading control.
