Fig. 3

CAG enhances H. pylori–induced autophagy. a AGS cells were infected with H. pylori 26695 or ΔCGT (MOI: 100) for 6 h. The protein levels of LC3B-II and β-actin were measured by immunoblotting. Cells treated with rapamycin (100 nM) were used as the positive control. b AGS cells were treated either with DMSO or CAG for 1 h and infected with H. pylori as described in (a). The samples were then subjected to immunoblotting for LC3B-II and β-actin. The autophagy response is shown by the LC3B-II/β-actin ratio. c GES-1 cells were treated with DMSO or CAG for 1 h and infected with H. pylori as described in (b). The samples were then analyzed for the protein levels of LC3B-II and β-actin by immunoblotting. d AGS cells were transfected with the plasmid encoding EGFP-tagged LC3 and then infected with H. pylori 26695 or ΔCGT as described in (a). The samples were then examined for autophagosomes with confocal microscopy. Cell periphery are marked with dotted-lines according to bright-field microscopy. Scale bar: 5 μm. e The autophagosomes observed in (d) were quantified by counting EGFP-LC3-positive puncta from > 25 cells in each group. f AGS cells transfected with the plasmid encoding EGFP-tagged LC3 were treated with DMSO or CAG, infected with H. pylori 26695 or ΔCGT as described in panel (b), and then imaged with confocal microscopy. Cell periphery are marked with dotted-lines according to bright-field microscopy. Scale bar: 5 μm. g Quantification of autophagosomes in (f). Approximately 25 cells from each group were randomly selected and counted to quantify EGFP-LC3-positive puncta. h GES-1 cells transfected with the plasmid of GFP-tagged LC3 were treated with DMSO or CAG, and infected with H. pylori 26695 or ΔCGT as described in (b), and then imaged by confocal microscopy. Scale bar: 10 μm. i The number of autophagosomes in (h) were quantified. Randomly ~ 25 cells from each group were examined to quantitate GFP-LC3-positive puncta. j Stomach cryosections of H. pylori 26695- and ΔCGT-infected mice were stained with anti-LC3B antibody (green) to estimate the level of induced autophagy. k The number of LC3-puncta in (j) were quantified. Randomly captured five images from each group were analyzed to quantitate LC3-positive puncta. **p < 0.01, *p < 0.05 vs. the control (n ≅ 25). In panels (e, g and i), data represent the mean ± SEM (standard error of the mean)