Fig. 2.

Inhibition of tubule formation in late endososmes/lysosomes causes the accumulation of cholesterol. a Western blot showing the downregulation of clathrin heavy chain (CHC) in wild-type mouse embryonic fibroblasts transfected with siRNA targeting CHC. b Quantification of the number of LAMP1-positive tubules in wild-type fibroblasts transfected with a control siRNA or a siRNA that downregulates CHC and expressing LAMP1 fused to mCherry, analyzed by live imaging. The graph shows the mean ± SEM. N > 58 cells analyzed in three independent experiments. T-test: ***p = 0.0004. c Quantification of the amount of filipin staining colocalized with the LAMP1 marker in fibroblasts transfected with a control siRNA or a siRNA that downregulates CHC. Downregulation of CHC resulted in a higher amount of cholesterol in late endosomes/lysosomes. The graph shows the mean ± SEM. N > 78 cells analyzed in three independent experiments. T-test: ***p = 0.0002. d Two-hour treatment of fibroblasts with the dynamin inhibitor dynasore (40 µM) induces the accumulation of cholesterol in Spg11^+/+ but not Spg11^−/− fibroblasts. The graph shows the mean ± SEM. N > 78 cells analyzed in three independent experiments. Two-way ANOVA: *p = 0.037, **p = 0.0098. e Live imaging of fibroblasts expressing LAMP1-mCherry and loaded with fluorescent cholesterol coupled to LDL. Note the presence of fluorescent cholesterol in tubules emanating from LAMP1-positive late endosomes/lysosomes (asterisk). Arrowheads point to a lysosomal tubule undergoing fission. Scale bar: 2 µm