Figure 2: ORP1L senses cholesterol to control autophagosome positioning.
(a) MelJuSo cells transfected with siCtrl or siORP1L were either or not cultured under lipid-depleted conditions and stained for LC3 and DAPI. Scale bar, 10 μm. Top right: quantification of scattering of LC3 structures under lipid-depleted conditions, over >100 cells were counted per experiment, bars indicate mean+s.d. from three experiments. Means were compared using Student’s t-test. Lower right: western blot to validate silencing of ORP1L. (b) Overview of the ORP1L constructs and their domains used in this study. The C-terminal ORD domain binds oxysterols. (c) MelJuSo cells transfected with the indicated GFP-tagged constructs were fixed and stained for LC3 and DAPI. Scale bar, 10 μm. (d) Quantification of cells with scattered autophagic vesicles in cells under different cholesterol manipulation conditions and expressing different GFP-tagged constructs, as shown in d and Supplementary Fig. 2b. Quantification was performed on >50 transfected cells per experiment, bars indicate mean+s.d. from independent triplicates. Significance calculated using Student’s t-test. (e) Superresolution microscopy on cells expressing HA-ORP1LΔORD, stained for HA and LC3, as indicated. Scale bar, 500 nm (f) Cryo-immuno-EM on HeLa cells expressing HA-LC3 and GFP-ORP1LΔORD stained with HA10 nm and GFP15 nm gold antibodies, respectively. Scale bar, 100 nm. (g) MelJuSo cells expressing GFP-ORP1LΔORD were fixed and stained for Rab7 and LC3. Scale bar, 10 μm. *P<0.05, **P<0.01, ***P<0.001. AV, autophagic vacuole.
