S. flexneri–induced B cell apoptosis is dependent on the virulence factor IpaD. (A) In vitro infections of the CL-01 B cell line with WT, mxiE, spa15, ipaC, ipaD, and complemented ipaD (ipaD/pIpaD) S. flexneri strains. Fold changes in cell number and percentages of PI⁺ cells are presented for infection with each strain over infection with the T3SA⁻ strain. Asterisks indicate statistical differences to T3SA⁻ bacteria. (B) CL-01 cell number and percentages of AnnV⁺PI⁻ (Annexin V⁺PI⁻) and PI⁺ populations at 24 and 48 h p.i. for uninfected cells incubated with 25 µg/ml His-tagged IpaD protein (UI + IpaD), T3SA⁻-infected cells, cells infected with T3SA⁻ and co-incubated with IpaD (T3SA⁻ + IpaD), and WT infected cells. Data are presented as fold change over the uninfected control. Asterisks indicate statistically significant differences between T3SA⁻ and T3SA⁻ + IpaD. (C) Percentages of apoptotic AnnV⁺PI⁻ cells after 24 h of co-incubation with T3SA⁻ and different concentrations of IpaD. Asterisks indicate statistical differences to T3SA⁻ bacteria alone. Three independent experiments were performed in triplicate for each condition/bacterial strain and data are presented as mean ± SEM. Statistically significant differences were determined by one-way (C) or two-way (A and B) ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.