Figure 1.
Effect of NH4Cl on VacA-induced vacuolation and apoptosis.
(A) Cells were treated with either vehicle (control), 5 mM NH4Cl, 120 nM VacA, or 5 mM NH4Cl plus 120 nM VacA for 6 hr. Vacuolating activity was determined by neutral red uptake assay. The data represent the mean ± SEM for 4 independent experiments. Data was assessed by ANOVA followed by Bonferroni method. *p<0.01, control vs VacA-treated cells. **p<0.01,Vac A alone vs NH4Cl plus VacA-treated cells. # p<0.01, control vs NH4Cl plus VacA-treated cells. (B) Cells were treated with either vehicle (control), 5 mM NH4Cl, 120 nM VacA, or 5 mM NH4Cl plus 120 nM VacA for 24 hr. Apoptosis was assessed by morphological criteria after DAPI staining. The data represent the mean ± SEM for 3 independent experiments. Data was assessed by one-way ANOVA followed by Bonferroni method. *p<0.05, control vs VacA-treated cells. **p<0.01, VacA alone vs NH4Cl plus VacA-treated cells. # p<0.01, control vs NH4Cl plus VacA-treated cells.
Figure 2.
VacA induces CHOP expression and phosphorylation of eIF2-α.
Cells were treated with either vehicle (control), 5 mM NH4Cl, 120 nM VacA, or 5 mM NH4Cl plus 120 nM VacA for 8 hr. (A) Nuclear extracts were obtained and resolved by SDS-PAGE. Proteins were identified by immunoblot analysis using anti-CHOP antibodies. Lamin B was used as a loading control. (B) The collected cells were subjected to real-time PCR. Relative mRNA fold induction was determined by normalization to 18S RNA. The data represent the mean ± SEM of 3 independent experiments, each performed in triplicate. Data was assessed by one-way ANOVA followed by Bonferroni method. *p<0.05, control vs VacA plus NH4Cl-treated cells. (C) Whole cell lysates were obtained and resolved by SDS-PAGE. Proteins were identified by immunoblot analysis using anti-phospho- and total- eIF2-α antibodies.
Figure 3.
VacA treatment induces expression of GRP78 mRNA.
Cells were treated with either vehicle (control), 5 mM NH4Cl, 120 nM VacA, or 5 mM NH4Cl plus 120 nM VacA for 8 hr. Expression of GRP78 was quantified by real-time PCR. The data represent the mean ± SEM for n = 3 studies. Data was assessed by one-way ANOVA followed by Bonferroni method. *p<0.05, control vs VacA-treated cells.
Figure 4.
CHOP contributes to VacA-induced apoptosis.
Cells were treated with either non-specific siRNA or CHOP siRNA for 48 hr. (A) CHOP mRNA expression was assessed by real-time PCR. Differences between groups were compared by using an unpaired two-tailed t-test. *p<0.05, non-specific siRNA-transfected cells vs CHOP siRNA-transfected cells. The data represent the mean ± SEM for n = 3 studies. (B) Cells were treated with either vehicle (control), 5 mM NH4Cl, 120 nM VacA, or 5 mM NH4Cl plus 120 nM VacA for 24 hr. Apoptosis was assessed by morphological changes after 30 min of DAPI staining. The data represent the mean ± SEM for n = 3 studies each performed in triplicate. Data was assessed by two-way ANOVA followed by Bonferroni method. *p<0.05, non-specific siRNA-transfected cells vs CHOP siRNA-transfected cells in VacA-treated cells. **p<0.01, non-specific siRNA-transfected cells vs CHOP siRNA-transfected cells in NH4Cl plus VacA-treated cells. (C) Cells were treated as indicated above. Cleaved PARP was assessed by immunoblotting. (D) After incubation with 5 mM NH4Cl plus 120 nM VacA for 24 hr, cells were collected and cleaved PARP was assessed by immunoblotting. Densitometry was performed and differences between groups were compared by using an unpaired two-tailed t-test.*p<0.05, non-specific siRNA vs CHOP siRNA transfected cells. The data represent the mean ± SEM for n = 3 studies.
Figure 5.
PERK contributes to CHOP activation and cell death.
Cells were treated with either non-specific siRNA or PERK siRNA for 48 hr. (A) After treatment with either vehicle (control), 5 mM NH4Cl, 120 nM VacA, or 5 mM NH4Cl plus 120 nM VacA for 8 hr, PERK protein expression was assessed by immunoblotting. (B) After treatment with either vehicle (control), 5 mM NH4Cl, 120 nM VacA, or 5 mM NH4Cl plus 120 nM VacA for 8 hr, cells were lysed and were subjected to immunoblot analysis with anti-phospho-eIF2-α and anti-total eIF2-α antibodies. (C) After incubation with 5 mM NH4Cl plus 120 nM VacA for 8 hr, cells were collected and real-time PCR was performed to assess CHOP mRNA expression. The data represent the mean ± SEM for n = 3 studies. Differences between groups were compared by using an unpaired two-tailed t-test. *p<0.05 non-specific siRNA vs PERK siRNA. (D) Cells were treated with either vehicle (control), 5 mM NH4Cl, 120 nM VacA, or 5 mM NH4Cl plus 120 nM VacA for 24 hr. Apoptosis was assessed by morphological changes after 30 min of DAPI staining. Data was assessed by two-way ANOVA followed by Bonferroni method. *p<0.01, non-specific siRNA-transfected cells vs PERK siRNA-transfected cells. The data represent the mean ± SEM for n = 3 studies. (E) Cells were treated as indicated above. Cleaved PARP was assessed by immunoblotting. (F) After incubation with 5 mM NH4Cl plus 120 nM VacA for 24 hr, Expression of Cleaved PARP was assessed by immunoblotting followed by densitometry. Differences between groups were compared by using an unpaired two-tailed t-test. *p<0.05, non-specific siRNA vs PERK siRNA transfected cells. The data represent the mean ± SEM for n = 3 studies. (G) Cells were examined by immunofluorescence microscopy for Bax following the treatment with vehicle (control) or 5 mM NH4Cl plus 120 nM VacA for 16 hr. The primary antibody used for the study recognizes the N-terminal region of Bax, which is exposed upon activation. Green fluorescence shows activated Bax, whereas blue fluorescence indicates DAPI staining of nucleus. Data represent the results of 3 independent experiments.
Figure 6.
VacA induces expression of Bim via activation of ER stress.
(A) Immunoblot analysis was employed for the assessment of pro-apoptotic-BH3-only proteins and Bcl-2 family proteins after the cells were treated with either vehicle (control), 5 mM NH4Cl, 120 nM VacA, or 5 mM NH4Cl plus 120 nM VacA for 8 hr. (B) Density of bands was analyzed to confirm increased expression of Bim protein in NH4Cl plus VacA-treated cells compared to control. Data was assessed by one-way ANOVA followed by Bonferroni method.*p<0.05, control vs VacA plus NH4Cl-treated cells. The data represent the mean ± SEM for n = 3 studies. (C) Cells were treated with either vehicle (control), 5 mM NH4Cl, 120 nM VacA, or 5 mM NH4Cl plus 120 nM VacA for 8 hr. Bim mRNA expression was assessed by real-time PCR. Data was assessed by one-way ANOVA followed by Bonferroni method. *p<0.05, control vs NH4Cl plus VacA-treated cells. The data represent the mean ± SEM for n = 3 studies. (D) After incubation with PERK siRNA or non-specific siRNA for 48 hr, cells were treated as above and were subjected for immunoblot analysis for Bim and actin. (E) After incubation with PERK siRNA or non-specific siRNA for 48 hr, cells were incubated with 5 mM NH4Cl plus 120 nM VacA for 8 hr. Expression of Bim was assessed by immunoblotting followed by densitometry. Differences between groups were compared by using an unpaired two-tailed t-test.*p<0.05, non-specific siRNA-transfected cells vs PERK siRNA-transfected cells. The data represent the mean ± SEM for n = 3 studies. (F) Cells were treated with either non-specific siRNA or PERK siRNA for 48 hr. After incubation with 5 mM NH4Cl plus 120 nM VacA for 8 hr, cells were collected and real-time PCR was performed to assess Bim mRNA expression. The data represent the mean ± SEM for n = 3 studies. Differences between groups were compared by using an unpaired two-tailed t-test. *p<0.05 non-specific siRNA vs PERK siRNA. Data represent the results of 3 independent experiments.
Figure 7.
ER stress markers and BH3-only proteins are elevated in human H. pylori-infected mucosa.
(A) mRNA was extracted from the biopsy specimen of H. pylori-negative and -positive gastric mucosa. Real-time PCR to quantify GRP78 mRNA expression was performed on the samples. Differences between groups were compared by using an unpaired two-tailed t-test. p<0.01, H. pylori-negative samples vs H. pylori-positive samples. H. pylori (−) n = 14, H. pylori (+) n = 22. (B) Immunohistochemistry of GRP78 in human gastric mucosa. GRP78 is identified by brown peroxidase staining. Right panel shows H. pylori-infected gastric mucosa (strong-positive GRP78 signal throughout the epithelium) and left panel shows healthy H. pylori-negative gastric mucosa (a lower GRP78 signal). Original magnification was 40×. (C) mRNA was extracted from the biopsy specimen of H. pylori-negative and -positive gastric mucosa. Real-time PCR of Bim (left panel) and PUMA (right panel) was performed on the samples. Differences between groups were compared by using an unpaired two-tailed t-test. H. pylori (−) n = 14, H. pylori (+) n = 22, *p<0.001, **p<0.05, H. pylori negative samples vs H. pylori positive samples.