Fig. 6

U18666A and bafilomycin A1 impairs Syntaxin17 trafficking to autophagosomes. A RPE1 cells were treated with U18666A (2 μg), chloroquine (5 μM), or bafilomycin A1 (25 nM) for 24 h in a serum-free medium. Cells were then subjected to lysotracker staining. Scale bar, 10 μm. B Intensities from 50 cells were acquired for each experimental group using ImageJ software. The bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. C Cells were transfected with GFP-LC3 for 12 h in serum-supplemented media. Cells were then treated with U18666A (2 μg), chloroquine (5 μM), or bafilomycin A1 (25 nM) for 4 h in a serum-starvation medium followed by 4% paraformaldehyde fixation and immunostaining with STX17 antibody. Scale bar, 10 μm. Arrowheads represent STX17 trafficking to autophagic structures (GFP-LC3). D Quantification of GFP-LC3 from 50 transfected cells was analyzed for each experimental group. The bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. E Determination of the number of STX17 positive GFP-LC3 puncta. Fifty transfected cells were analyzed for each experimental group. The bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. F Percentage of STX17 positive GFP-LC3 puncta were calculated from D and E. The bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. G Cells were treated with bafilomycin A1 (25 nM) for different time points in serum-starvation medium. Cells were then subjected to Lysotracker staining. Scale bar, 10 μm. H Lysotracker intensities from 50 cells were acquired using the ImageJ software for each experimental group. The bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test