Fig. 2

Autophagy impairment by U18666A significantly correlates with lysosomal cholesterol sequestration. A RPE1 cells were treated with either U18666A (2 μg) or chloroquine (5 μM) in a serum-starvation medium for different durations. Cells were then subjected to filipin staining. Scale bar, 10 μm. B Fluorescence intensity of filipin was measured using ImageJ software. Filipin intensity was compared for different time points in the presence of U18666A (2 μg) or chloroquine (5 μM). Filipin intensity from 50 cells was acquired for each experimental group. The bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. C Cells treated with either U18666A (2 μg) or chloroquine (5 μM) in a serum-starvation medium for 4 h or 8 h. Cells were then subjected to Western blot for LC3 and ACTB. D Intensity of the bands from C was quantified using Image J software, and the LC3-II/ACTB ratio was measured. Bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. E, F Pearson’s correlation coefficient for the association between the impaired autophagy flux and cholesterol accumulation between 4 and 8 h in the presence of U18666A or chloroquine was determined by plotting individual LC3-II/ACTB ratio and filipin intensity from three different experiments