ED Fig. 7. VDIMs are not multivesicular bodies (MVB).

a, Representative images showing mitotracker (magenta), TOM20 (cyan), LBPA (grey) in cells expressing LAMP1-GFP (green). Bottom: Higher magnification of indicated regions. Arrowheads indicate VDIMs lacking LBPA. Arrow indicates VDIM positive for LBPA. b, Number of VDIMs positive for LAMP1 or LBPA from experiments as in (a) (n=243 vesicles, 30 cells, 3 experiments). c, Representative images showing mitotracker (magenta), TOM20 (cyan), CD63 (grey) in cells expressing LAMP1-GFP (green). Bottom: Higher magnification of indicated regions. Arrowheads indicate VDIMs lacking CD63. Arrow indicates VDIM positive for CD63. d, Number of VDIMs positive for LAMP1 or CD63 from experiments as in (c) (n=167 vesicles, 17 cells, 2 experiments). e, Schematic illustrating lysosome and MVB fusion regulated by Arl8b GTPase. f, Localization of Lamp1 (magenta) and LBPA (green) in cells expressing GFP-Arl8b-WT (blue) or GFP-Arl8b-DN (blue). Right: Pixel intensity plots for dashed line. Arrows indicate LBPA negative lysosomes in cells expressing GFP-Arl8b-DN. g, VDIM formation in cells expressing GFP-Arl8b-WT (green) or GFP-Arl8b-DN (green). Higher magnification of indicated regions are shown where arrowheads indicate the VDIM. h, Number of VDIMs in cells expressing Arl8b-WT or Arl8b-DN in experiments as in (g) (n= 30 cells, 3 experiments). i, Number of LAMP1 positive VDIMs in cells expressing Arl8b-WT or Arl8b-DN in experiments as in (g) (n=30 cells, 3 experiments). Data shown are mean±SEM. Statistical significance was calculated using two-tailed Student’s unpaired t-test. P values calculated are indicated. Scale bars: main panels 10μm, magnified panels 3μm.