Fig. 4

Genetic ablation of autophagy does not induce lysosomal cholesterol accumulation. A WT and ATG5 KO MEFs were incubated in either a serum-supplemented medium or serum-starvation medium for 24 h. Cells were then subjected to Western blot for LC3, ATG5, and ACTB. B MEFs were incubated in either a serum-supplemented medium or serum-starvation medium for 24 h. Cells were then subjected to filipin staining. Scale bar, 10 μm. C Fluorescence intensity of filipin was measured using ImageJ software. 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. D RPE1 cells were transfected with control siRNA or ATG7 siRNAs for 48 h followed by further incubation in serum-starvation medium for an additional 12 h. Cells were subjected to Western blot for LC3, ATG7, and ACTB. E, F Intensity of the bands from D was quantified using Image J software. LC3-II/ACTB ratio E and ATG7/ACTB ratio F were determined. Bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. G RPE1 cells were transfected with control siRNA or ATG7 siRNAs for 48 h followed by further incubation in serum-starvation medium for an additional 12 h. Cells were subjected to filipin staining. Scale bar, 10 μm. H Fluorescence intensity of filipin was measured using the ImageJ software. 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