Fig. 1

U18666A and chloroquine impair autophagy through different mechanisms. A RPE1 cells treated with different concentrations of U18666A in a serum-starvation medium for 24 h. Cells were then subjected to Western blot for LC3 and ACTB. B Cells treated with different concentrations of chloroquine in a serum-starvation medium for 24 h. Cells were then subjected to Western blot for LC3 and ACTB. C Cells were maintained with U18666A (2 μg), chloroquine (5 μM), or combined treatment with both drugs in a serum-starvation for 24 h. Cells were then subjected to Western blot for LC3, SQSTM1, and ACTB. D RPE1-mRFP-GFP-LC3 cells treated as in C, fixed with 4% paraformaldehyde, and stained with DAPI. Scale bar, 10 μm. E, F, Autophagy status is determined by counting autophagosomes (yellow puncta with both GFP and RFP signal) or 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