Mito-VPS41 does not recruit LC3, CI-M6PR, AP3, or endo/lysosomal tracers. (A) Confocal images of endogenous LC3. HeLa^VPS41KO cells expressing mito-VPS41^WT-GFP and Arl8b-HA show no recruitment of LC3 to mitochondria. (B) Immuno-EM of HeLa^VPS41KO cells expressing mito-VPS41^WT-V5 and Arl8b-HA and immunolabeled for endogenous Tom20 (10-nm gold). Vesicles accumulating around mitochondria (arrows) are negative for Tom20. For original EM images, see Fig. S3 D. (C) Confocal image of endocytosed dextran (2 h) in HeLa^VPS41KO expressing Arl8b-GFP and mito-VPS41^WT-V5 and immunolabeled for V5 (10-nm gold). There is no recruitment of dextran to mito-VPS41^WT-V5. (D) Immuno-EM of HeLa^VPS41KO cells expressing mito-VPS41^WT-V5 and Arl8b-HA, incubated with BSA⁵, and labeled for HA and V5 (10- and 15-nm gold, respectively). Arl8b-HA–positive vesicles (blue) are devoid of endocytosed BSA⁵. E, endosome (yellow); M, mitochondria (purple). Scale bars: 500 nm. For original EM figures, see Fig. S3 D. (E) Confocal images showing CI-M6PR distribution in HeLa^VPS41KO cells expressing mito-VPS41^WT-GFP and Arl8b-HA. (F) Quantification of CI-M6PR fluorescence on mitochondria shows similar distributions in cells expressing mito-VPS41^WT-GFP or negative control mito-GFP (see also Fig. S3 E). Data represent means ± SEM. n > 25. Statistical significance was assessed using an unpaired t test, ns: not significant. (G) Confocal images of HeLa^VPS41KO cells expressing Arl8b-HA and mito-VPS41^WT-GFP and incubated with LysoTracker (30 min) to visualize acidic compartments. (H) Quantification of overlap between LysoTracker and mitochondria shows no effect of mito-VPS41^WT-GFP expression (see also Fig. S3 F). Data represent means ± SEM. n > 20. Significance was obtained using an unpaired t test, ns: not significant. (I) Confocal images showing endogenous localization of AP3D in PC12^VPS41KO cells transfected with Arl8b-mCherry and mito-VPS41^WT-GFP. (J) Quantification of overlap between AP3D and mitochondria shows no effect of mito-VPS41^WT-GFP (see also Fig. S3, G and H). Scatter plot represents the mean ± SEM. n ≥24. Statistical analysis was obtained using an unpaired t test, ns: not significant. Scale bars in I are 5 µm. Scale bars in A, C, E, and G are 10 µm and 5 µm (inset).