Puma-deficient B cells were more resistant than wild-type B cells to apoptosis on in vitro activation. (A-B) Wild-type and Puma-deficient (Puma−/−) splenic B cells (2 × 106 cells/mL) were stimulated for 8 days in culture by the combination of Fab'2 anti–mouse μ antibody fragments (10 μg/mL) plus anti–mouse CD40 antibodies (5 μg/mL; A) or LPS (10 μg/mL; B). Activated B cells were quantified by flow cytometry (see Figure 3C) from day 2 to day 8. Data represent the mean (± SEM) of n = 6 mice for each genotype. (*P < .05, **P < .01). Expression levels of Puma, Mcl-1, and GAPDH (loading control) proteins were determined by immunoblotting. (C) Wild-type (Wt) and Puma-deficient (Puma−/−) splenic B cells (2 × 106 cells/mL) were stimulated for 6 days by the combination of Fab'2 anti–mouse μ antibody fragments (10 μg/mL) plus anti–mouse CD40 antibodies (5 μg/mL) or LPS (10 μg/mL). The percentages of apoptotic cells were assessed by flow cytometry. Data represent mean (± SEM) of n = 5 mice per genotype. (**P < .01). (D) Wild-type (Wt) and Puma-deficient (Puma−/−) B cells were labeled with CFSE and then stimulated in culture with Fab'2 anti–mouse μ antibody fragments (10 μg/mL) plus anti–mouse CD40 antibodies (5 μg/mL). B-cell proliferation (reflected by dilution of CFSE fluorescence intensity) was assessed by FACS analysis at days 0 and 4 of activation. Results shown are representative of 3 independent experiments.
