Fig. 3

Induction of lysosomal cholesterol sequestration by bafilomycin A1 significantly correlates with autophagy impairment. A Cells were treated with different concentrations of bafilomycin A1 in a serum-starvation medium for 24 h. Cells were then subjected to Western blot for LC3 and ACTB. B Cells were treated with bafilomycin A1 (25 nM) in a serum-starvation medium for 4 h and 8 h. Cells were then subjected to Western blot for LC3 and ACTB. C Intensity of the bands from B was quantified using Image J software, and the LC3-II/ACTB ratio was determined. Bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. D Cells were treated with bafilomycin A1 (25 nM) in a serum-starvation medium for a different duration. Cells were then subjected to filipin staining. Scale bar, 10 μm. E Fluorescence intensity of filipin was measured using ImageJ software. Bar graph represents the mean ± SD (n = 3 experiments). Filipin intensity from 50 cells was acquired for each experimental group. Bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. F Pearson’s correlation coefficient for the association between impaired autophagy flux and cholesterol accumulation between 4 and 8 h in the presence of bafilomycin A1 was determined by plotting individual LC3-II/ACTB ratio and filipin intensity from three different experiments