Overexpression of CAPZA1 inhibits the formation of CagA-degrading autolysosomes via repressing LAMP1 expression. (a) AGS cells were transfected with pCMV-control or pCMV-CAPZA1 plasmid, incubated for the indicated time periods, and then CAPZA1 and LAMP1 expression levels were determined. (b) AGS cells were transfected with pCMV-CAPZA1 plasmid, infected with H. pylori for 5 h (MOI 50), and incubated in a medium containing antibiotic for 24 h. Then, LAMP1 and phalloidin staining were performed. Nuclei (blue) were stained with DAPI. Scale bar: 20 μm. The number of LAMP1-staining puncta were counted by using the ImageJ program. Data are presented as the mean ± SD of 3 independent images. NS, not significant. (c) AGS cells were transfected with pCMV-CAPZA1 plasmid, infected with H. pylori for 5 h (at MOI 50), and incubated in a medium containing antibiotic for 24 h. Then, LysoTracker Red DND-99 staining was performed. Nuclei (blue) were stained with DAPI. Scale bar: 20 μm. The number of LysoTracker Red-staining puncta were counted by using the ImageJ program. Data are presented as the mean ± SD of 3 independent images. NS, not significant. (d) AGS cells were transfected with pCMV-control or pCMV-CAPZA1 plasmids, infected with H. pylori for 5 h (MOI 50), and incubated in a medium containing antibiotic for the indicated times. Data are presented as the mean ± SD of 3 independent assays. *P < 0.05, **P < 0.01. (e) AGS cells were transfected with pCMV-CAPZA1 plasmid, infected with H. pylori for 5 h (at MOI 50), and incubated in a medium containing antibiotic for 24 h. Then, a ChIP assay was performed on these cells with an anti-LRP1-carboxyterminal end antibody. Real-time PCR demonstrated relative enrichment of LAMP1 target promoter genes in the DNA fragments pulled down by an anti-LRP1-carboxyterminal end antibody. Result shown is representative of those observed in 2 independent experiments. (f) AGS cells were infected with H. pylori for 5 h (MOI 50) and incubated in a medium containing antibiotic for 24 h. Then, a ChIP assay was performed on these cells with an anti-CAPZA1 antibody. Real-time PCR demonstrated relative enrichment of LAMP1 target promoter genes in the DNA fragments pulled down by an anti-CAPZA1 antibody. Result shown is representative of those observed in 2 independent experiments.