PINK1 in mitochondrial function and EV production. (a) MDA-MB-231 cells were transfected with siRNAs targeting PINK1 (siPINK1) or a nontargeting control (siNT). Transfected cells were treated with the indicated concentrations of CCCP for 3 h, and PINK1 levels were determined using Western blotting with actin as a loading control. (b) MDA-MB-231 cells were transfected siRNAs targeting PINK1 (siPINK1) or nontargeting control (siNT) and treated with CCCP for 3 h before being analyzed for total Ub and phosphorylated Ub by Western blotting. Actin was used as a loading control. (c) MDA-MB-231 cells were transfected with siRNAs targeting PINK1 (siPINK1) or nontargeting (NT) siRNA. 5 d following transfection, oxygen consumption was determined using the Seahorse XF^e96 Extracellular Flux Analyzer. Sequential treatment with oligomycin, FCCP, and rotenone/antimycin A (Rot+AntA) was performed as indicated. Maximum OCR was determined by subtracting rotenone/antimycin A OCR from basal OCR values. Values represent the mean of three independent experiments; error bars signify the SEM. **, P < 0.01; ***, P < 0.001; paired Student’s t test. (d) Conditioned medium was collected from cells isolated from an MMTV-PyMT–driven mammary carcinoma in which PINK1 expression had been disrupted by CRISPR using two distinct guide sequences (PINK1 no. 1 and PINK1 no. 2) or a nontargeting guide sequence (NT), and EVs were purified from these by differential centrifugation. EVs were analyzed using nanoparticle tracking and Western blotting for CD63 as in Fig. 1 a. Values represent the mean of three independent experiments; dotted line and error bars represent SEM. ***, P < 0.001; t test with Welch’s correction.