Fig. 9

Methyl- β-cyclodextrin efficiently rescues impaired autophagy flux in the presence of U18666A. A Cells were treated with U18666A (2 μg), chloroquine (5 μM), or bafilomycin A1 (25 nM) for 12 h in a serum-starvation medium. Cells were further incubated in the presence or absence of MβCD (20 μM) for an additional 24 h without changing medium and subjected to Western blot for LC3, SQSTM1, and ACTB. B Intensity of the bands from A was quantified using the ImageJ software, and the SQSTM1/ACTB ratio was determined. Bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. C RPE1-mRFP-GFP-LC3 cells were treated as in A, fixed with 4% paraformaldehyde, and stained with DAPI. Scale bar, 10 μm. D, E, Autophagy status was determined by counting autolysosomes (punctate structures with only RFP signal) or autophagosomes (yellow puncta with both GFP and RFP signal). At least 100 cells were counted for each experimental group. Bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. F RPE1-mRFP-GFP-LC3 cells were co-treated with U18666A (2 μg) and chloroquine (5 μM) for 12 h in a serum-starvation medium. Cells were further incubated in the presence or absence of MβCD (20 μM) for an additional 24 h without changing medium, fixed with 4% paraformaldehyde, and stained with DAPI. Scale bar, 10 μm. G Autophagy status was determined by counting autolysosomes (punctate structures with only RFP signal). At least 100 cells were counted for each experimental group. The bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test