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Fig. 4 | Journal of Biomedical Science

Fig. 4

From: Enhanced enzymatic production of cholesteryl 6ʹ-acylglucoside impairs lysosomal degradation for the intracellular survival of Helicobacter pylori

Fig. 4

CAG impairs autophagosomal degradation. a AGS cells transfected with the plasmid encoding EGFP-tagged LC3 were treated with DMSO or CAG, infected with H. pylori 26695 or ΔCGT for 6 h, either in the absence or presence of bafilomycin A1 (BafA1, 10 nM), fixed, and imaged with confocal microscopy. Scale bar: 5 μm. b More than 50 cells in (a) that were positive for EGFP were randomly selected and counted to quantify the EGFP-positive autophagosome puncta. c GES-1 cells transfected with the plasmid of EGFP-tagged LC3 were treated, infected, and imaged as described in (a). d Randomly selected EGFP-positive cells were quantified for EGFP-positive autophagosome puncta. e AGS cells treated and infected as mentioned in (a) were subjected to immunoblotting for LC3B-II and β-actin. f GES-1 cells treated and infected as mentioned in (a) were examined for the protein levels of LC3B-II and β-actin by Western blotting. g CAG disrupts autophagy flux. AGS cells were transfected with the mRFP-GFP-LC3 tandem plasmid and then treated with DMSO or CAG, infected with H. pylori 26695 or ΔCGT for 6 h, and then imaged with confocal microscopy. Scale bar: 5 μm. h More than 50 cells from each group of (g) were randomly selected and counted to statistically analyze autophagy flux, shown as the yellow/red ratio. Yellow signal resulted from the puncta that are GFP+RFP+, whereas red signal originated from the RFP+ puncta only. i AGS cells were co-transfected with plasmids encoding EGFP-LC3 and TPC2-RFP, treated with CAG, and infected as indicated in (a). The samples were then imaged with confocal microscopy. Scale bar: 5 μm. j More than 20 cells that were randomly selected for quantification of EGFP and RFP fluorescence. The co-localization coefficient (signals that were positive for both TPC2-RFP and EGFP-LC3) was quantified using image J software. The data presented in panels (b, d, h and j) represent the mean ± SEM. **p < 0.01, *p < 0.05 vs. the control; n.s. (not significant)

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