Cholesterol accumulation inhibits lysosomal fusion.Figure 3Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar: 10 μm (A, C, D, F). Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar: 10 μm (A, C, D, F). Cholesterol accumulation inhibits lysosomal fusion. Endolysosomal membrane cholesterol measurements and Filipin staining were carried out in either (A) WT MEFs loaded with cholesterol or in MSD and (D) MPS‐IIIA MEFs treated with MβCD. Arrowheads and enlarged images show cholesterol accumulation in endolysosomes of cholesterol‐loaded WT MEFs. After treatments the rate of autophagosome maturation (B, E) and the transport of fluorescent dextran to lysosomes (C, F) were also analysed as in Figure 1. WT controls for autophagosome maturation and dextran transport experiments were performed as shown in Figure 1. (A-F) Values represent the mean±s.e.m. values of three independent experiments. *P0.05, Student's t‐test: (A, C): WT versus WT+cholesterol; (B): WT versus WT+cholesterol for each time point; (D, F): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD; (E): MSD versus MSD+MβCD and MPS‐IIIA versus MPS‐IIIA+MβCD for each time point. Scale bar: 10 μm (A, C, D, F).