Supplementary Figure 3: Cholesterol is necessary for migrasome formation.

a, Tspan1-mCherry-, Tspan7-mCherry-, Tspan9-mCherry- and CD82-mCherry-expressing NRK cells were observed by confocal microscopy. Enrichment of Tspan proteins on migrasomes is revealed by heat map images. Scale bars, 10 µm. b, NRK cells stably expressing Tspan4-GFP were treated by MβCD (Methyl-β-cyclodextrin, 10 mM) for 0 min and 30 min. Confocal images were collected at each time point. Scale bar, 10 µm. c, Statistical analysis of migrasome numbers at each time point in images like those in (b). Data shown represent mean±SEM; n=84 cells for MβCD 0 min; n=109 cells for MβCD 30 min. ***P<0.001. d, Relative total cellular cholesterol level of cells grown under different cholesterol depletion conditions, including 10% full cholesterol medium (FBS), cholesterol depletion medium (LPDS) and cholesterol depletion medium with 30 μM Pravastatin. Data shown represent mean±SEM (n=3 independent experiments), ***P<0.001. Experiments related to a-d were independently repeated 3 times. The quantifications in c are pooled from three independent experiments. Two-tailed unpaired t-test method was used for statistical analysis. The statistical details for c and d were available in Supplementary Table 4.