Cholesterol-dependent MCSs between LEs and the ER allowing in trans VAP interactions with the Rab7–RILP complex. (A) Electron micrographs of multivesicular bodies in Mel JuSo cells expressing HA-VAP and different variants of GFP-ORP1L, as indicated. Sections were stained with anti-HA antibodies detected with 15-nm gold particles. The gold particles are highlighted by red dots, and the ER membrane (containing HA-VAP) is indicated by blue lines. The original images are shown in Fig. S5 C. (B) The percentage of multivesicular bodies contacting ER structures and the contact area between the ER and LE membranes was determined in Mel JuSo cells expressing the ORP1L variants as shown in A. Over 50 multivesicular bodies were considered per condition. The data on contact area from two independent quantifications were binned as indicated, and error bars show SEM. (C) Electron micrographs of multivesicular bodies in HA-VAP– and GFP-ORP1L–expressing MelJuSo cells exposed to different cholesterol-manipulating conditions, as indicated. Sections were stained with anti-HA antibodies marked by 15-nm gold particles. The gold particles are highlighted by red dots, and the ER membrane (containing HA-VAP) is indicated by blue lines. The original images are shown in Fig. S5 F. (D) The percentage of multivesicular bodies contacting ER structures and the contact area between the ER and LE membranes from MelJuSo cells expressing ORP1L under various conditions of cholesterol manipulation (as shown in C). Over 50 multivesicular bodies were considered per condition. The data on contact area from two independent quantifications were binned as indicated, and error bars show SEM. Bars, 200 nm.