A Type III Effector NleF from EHEC Inhibits Epithelial Inflammatory Cell Death by Targeting Caspase-4

Figure 3

EHEC ΔnleF-induced inflammatory cell death is mediated by caspase-4. (a–c) HT29 cells transfected with the indicated siRNAs were infected with EDL933, ΔnleF, or ΔnleF/nleF at a MOI of 100 and then incubated at 37°C for the indicated time periods. (a) Aliquots of supernatants were subjected to the cytotoxicity assay. The knockdown efficiency of siCASP1, siCASP4, or siCASP5 was analyzed by RT-PCR. (b) Infected cells were fixed at the indicated time points and then subjected to Giemsa staining. Red arrowheads indicate cells whose cytoplasm disappeared. The number of infected cells undergoing chromatin condensation was calculated from at least 300 cells and is shown in the lower panels. (c) Aliquots of supernatants were subjected to an IL-1β ELISA assay. (d) 293T cells were cotransfected with Myc-NleF and Flag-caspase-4 or Flag-caspase-1 or Flag-caspase-5 expression plasmids or Flag-vector; anti-Flag immunoprecipitates were analyzed by immunoblotting with anti-Myc or anti-Flag antibodies. Graphs show the means ± SEM of triplicate wells; all data are representative of three independent experiments. . values were calculated using two-tailed Student’s -test.

(a)

(b)

(c)

(d)