Fig. 2: Detection of caspase-activation and cell death upon infection with Helicobacter pylori.

a AGS cell lines were infected with different multiplicities of infection (MOI) of H. pylori G27 strain for 18 h. Active caspase3 was measured by flow cytometry using an antibody recognizing the cleavage product of caspase-3-activation in three independent experiments. b AGS cell lines were infected with different MOI of H. pylori G27 strain for 24 h. Epithelial cell survival was measured by LDH release assay in three independent experiments. Cytotoxicity was calculated as described in Methods. c AGS cell lines were infected with different MOI of H. pylori G27 strain. Cells were counted and 500 cells were seeded in triplicates in medium supplemented with antibiotics after 18 h of infection. Clonogenic growth was determined with crystal violet staining after one week. Data are from three independent experiments. Not normalized data are shown in S8D. d AGS cell lines were infected with H. pylori G27 strain at an MOI of 100 for 18 h. After 15 h, biotinylated VAD-fmk was added. Cells were lysed, and inhibitor-bound caspases were precipitated from cell lysates using neutravidin beads. Precipitated (i.e. inhibitor-bound, activated) caspase3 was detected by Western blotting. Shown is a representative Western blot from two independent experiments. e AGS cell lines were infected with various MOI of H. pylori G27 strain for 18 h. Effector caspase activity was measured in cell lysates with an AC-DEVD-AMC reporter substrate. Increasing fluorescence indicates effector caspase activity. Data are from three independent experiments. Not normalized data are shown in S8E. f KATOIII wild type cell lines were infected with different MOI of H. pylori G27 strain for 18 h. Active caspase3 was measured by flow cytometry using an antibody recognizing the cleavage product of caspase-3-activation (data are from three independent experiments). g KATOIII cell lines were infected with H. pylori G27 strain at an MOI of 10 for 5 h. Effector caspase activity was measured in cell lysates with an AC-DEVD-AMC reporter substrate. Increasing fluorescence was detected then effector caspases were active (data are from four independent experiments). Not normalized data are shown in S8F. h KATOIII wild type cell lines were infected with different MOI of H. pylori G27 strain for 18 h. Cell survival was measured by trypan blue exclusion in three independent experiments. Not normalized data are shown in S8G. i KATOIII wild type cell lines were infected with different MOI of H. pylori G27 strain for 18 h. Epithelial cell survival was measured by LDH release assay in three independent experiments. Not normalized data are shown in S8H. Data information: Bars represent the mean and dots the value of independent experiments. Error bars show standard error of mean. Ns: p > 0.05, *, p < 0.05, **, p < 0.01. Significance was tested by one sample T-Test (g, h, i) parametric (a, f: Dunnett´s post hoc test; c: Sidak ´s post hoc test) and non-parametric (e: Dunn´s post hoc test) one-Way ANOVA. CTRL, non-targeting control gRNA; Bax^−/−Bak^−/−, double knockout of Bax and Bak by CRISPR/Cas9. I, input; B, bound to beads; U, unbound.