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. 2015 Nov 23;211(4):913–931. doi: 10.1083/jcb.201502074

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© 2015 Ittig et al.

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Figure 4. — Delivery of human tBID into cells induces massive apoptosis. (a) Translocated tBID induces CASP3 cleavage. Cleavage of CASP3 into the p17 subunit was monitored by Western blot analysis of HeLa cells infected for 60 min with the indicated strains at an MOI of 100 or treated with staurosporine. (b) Translocated YopE_1–138-BID fusion proteins reach endogenous levels. Digitonin-lysed HeLa cells, infected for 1 h with the indicated strains as shown in panel a at an MOI of 100, were analyzed by Western blot analysis with an anti-BID antibody to compare endogenous BID, translocated YopE_1–138-BID and YopE_1-138-tBID levels. (c) Translocated tBID induces PARP cleavage. Cleavage of PARP was monitored by Western blot analysis of HeLa cells infected for 60 min with the indicated strains at an MOI of 100. (d) Delivery of t-BID activates CASP3 as monitored by measuring the fluorogenic substrate ac-DEVD-amc after incubation for 1 h with cell lysates of HeLa cells infected for 60 min with the indicated strains at an MOI of 100. Pan-caspase inhibitor z-VAD was added at 50 µM when indicated. Error bars indicate standard errors of the mean. (e) Translocated tBID induces massive cell loss. HeLa cells were infected with the indicated strains at an MOI of 100 for 1 h or treated with staurosporine. After fixation, cells were stained for nuclei (blue) and F-actin (gray). Bar, 50 µm. (f) Delivery of t-BID leads to a strong reduction in cell number, assessed by counting nuclei. HeLa cells were infected for 60 min with the indicated strains at an MOI of 100. Cells were preincubated 1 h before infection with the indicated concentrations of z-VAD. z-VAD blocks the effect of translocated t-BID in a dose-dependent manner. Automated image analysis was performed on n = 18 images per condition. Error bars indicate standard errors of the mean. Statistical analysis was performed using a Mann-Whitney test (***, P < 0.001; ****, P < 0.0001; ns, not significant). (g) Metabolic activity of HeLa cells 24 h after infection with the indicated strains at indicated MOIs assessed by resazurin assay. 1 h p.i. penicillin and streptomycin were added. Error bars indicate standard errors of the mean.