TMEM16F is involved in MVB formation upon TCR engagement. (A and B) Confocal microscopy analysis of LBPA staining of Jurkat cells seeded on coverslips coated with αCD45 (nonstimulatory) or αCD3 (stimulatory). 2-µm z stack of images is shown. Representative images are shown in A. Number of LBPA-positive vesicles was quantified in (B). Bars, 5 µm. DIC, differential interference contrast. n = 142 for αCD45; n = 190 for αCD3. (C) Quantification of LBPA-positive vesicles in nontarget (control) or T16F-KD (TMEM16F-knockdown) Jurkat T cells treated with αCD45 or αCD3. n = 33, 44, 44, and 85, from left to right. (D–F) Electron microscopy of MVBs in nontarget or T16F-KD Jurkat T cells. Representative electron micrographs are shown in D. Arrows indicate MVBs. Bars, 100 nm. Quantification of the number of ILVs per MVB and categorization of MVB stages are shown in E and F, respectively. n = 83 for nontarget and n = 71 for T16F-KD. Results are displayed as mean ± SEM. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant, using Student’s t test. Data are representative of three experiments.