Expression of Puma was up-regulated in activated human B cells. (A) Human purified tonsillar B lymphocytes (2 × 10⁶ cells/mL) were left untreated or stimulated with mitogenic doses of the S aureus Cowan 1 strain (pansorbin: SAC; 1/10 000) for 24 hours. Expression levels of various Bcl-2 family proteins and tubulin (loading control) were analyzed by immunoblotting. The percentages of apoptotic cells were determined by flow cytometric analysis. Data represent mean (± SEM; B). Human purified tonsillar B lymphocytes (2 × 10⁶ cells/mL) were stimulated with SAC (1/10 000) in the presence or absence of the broad-spectrum caspase inhibitor QVD-OPH (10nM) to prevent apoptosis. Expression levels of Puma and tubulin (loading control) were evaluated by immunoblotting. The percentages of apoptotic cells were determined by flow cytometric analysis. Data represent mean (± SEM). (C) Human purified tonsillar B lymphocytes (2 × 10⁶ cells/mL) were stimulated for 24 hours in the presence or absence of SAC (1/10 000), or anti–human μ antibodies (10 μg/mL) or anti–human CD40 antibodies (5 μg/mL) either by themselves or in combination. Expression levels of Puma and tubulin (loading control) were analyzed by immunoblotting. DNA synthesis was quantified by measuring incorporation of ³H-thymidine during the last 16 hours of culture. Results are representative of 4 independent experiments.