Figure 1: Cholesterol modulates autophagosome positioning.

(a) HeLa or MelJuSo cells were stained for LC3 and 4,6-diamidino-2-phenylindole (DAPI), or with filipin to detect cholesterol. Scale bar, 10 μm. Panels below: quantification for the presence of a detectable perinuclear LC3 cluster: >100 cells were analysed per experiment. For filipin labelling, signal intensity was measured for at least three fields per experiment and was normalized to signal intensity in HeLa cells, which was set at 1. Bars indicate mean+s.d. from independent triplicates, significance calculated using Student’s t-test. (b) MelJuSo cells cultured for 24 h under cholesterol-depleting conditions (see Methods) or exposed for 24 h to 3 μM U18666 were stained for LC3 and DAPI. Scale bar, 10 μm. Lower panel: quantification of the average distance (μm) of the AVs from the nucleus (using the LasAF software), from >10 cells per experiment. Bars indicate mean+s.d. from independent triplicates, significance calculated using Student’s t-test. (c) Distribution and dynamics of mCherry-LC3-marked vesicles in control versus lipid-depleted MelJuSo cells. Confocal image at start of time lapse is shown on the left, vesicle trajectories over a 480-s interval are displayed on the right. Colours of individual vesicle trajectories reflect maximal displacement rate (blue=0, red=0.8 μm s⁻¹) achieved during the 480-s interval. Scale bar, 10 μm. (d) Quantification of the localization of mCherry-LC3 vesicles either or not labelling for Lysotracker in MelJuSo cells treated as indicated. At least five cells per replicate were quantified, bars indicate mean+s.d. from independent triplicates. Significance calculated using Student’s t-test (Supplementary Movies 1–3). NS, not significant, *P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001). AV, autophagic vacuole.