Bacterial co-signals sensitize human B lymphocytes to IpaD-mediated mitochondrial apoptosis. (A) Apoptotic CL-01 B cells after incubation for 24 h with live, gentamicin-treated, sonicated, or heat-killed ipaD bacteria or a cocktail of LPS, PGN, and CpG (C). Percentages of AnnV⁺PI⁻ cells are shown, in the presence (+IpaD) or absence of IpaD. Asterisks indicate statistical differences to the uninfected control (on column) or between conditions (indicated by bars) determined by one-way ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01. (B) Loss of MMP as assessed by flow cytometry with the fluorescent probe JC-1. Fold changes in the percentage of cells that lost MMP are presented for T3SA⁻ and T3SA⁻ + IpaD over the uninfected control. Asterisks indicate statistical differences to the uninfected control determined by two-way ANOVA with Bonferroni post-test. **, P < 0.01; ***, P < 0.001. (C–F) Dynamic regulation of mitochondrial pro- and anti-apoptotic proteins over time. Protein amounts were assessed by Western blot at 1, 3, 5, and 24 h p.i., and are presented as fold change over the uninfected control for IpaD alone, T3SA⁻, and T3SA⁻ + IpaD after normalization to actin. (C and D) Protein amounts of pro-apoptotic Bad (C) and Bax (D). (E and F) Protein amounts of anti-apoptotic Bcl-2 (E) and Bcl-x_L (F). All data are presented as mean ± SEM for three independent experiments.