Expression of Puma in normal and malignant B cells was associated with a germinal center phenotype. (A) Expression of Bcl-2 (i), Puma (ii), Mcl-1 (iii), and Bcl-xL (iv) was assessed in histologic sections of human lymph nodes by in situ immunostaining (×100 magnification). As a control, the same tonsil sample was stained with normal rabbit serum (at a dilution 1/8000) using the same protocol that was used for detection of PUMA (data not shown). (B) Tonsilar B cells (2 × 106 cells/mL) were activated in culture with SAC (1/10 000) for 24 hours. Cells were lysed in CHAPS buffer and cell lysates (50 μg protein) were immuno-precipitated with antibodies against Puma or control Ig. Immune complexes as well as input from Puma immunoprecipitates were analyzed by immunoblotting with antibodies against Mcl-1, Bcl-xL, or Puma. The asterisk represents a nonspecific cross-reactive band. (C) Expression of Puma and tubulin (loading control) was analyzed by immunoblotting in 3 Burkitt lymphoma–derived cell lines (BJAB, BL41, and Ramos) and compared with the expression levels in purified human tonsillar B lymphocytes. (D) BL41 cells were cultured for 24 hours in presence of various doses of the c-Myc inhibitor 10058-F4. Expression levels of Puma, Bcl-2, Bcl-6, and GAPDH (loading control) were analyzed by immunoblotting. (E-F) BL41 cells (E) or Ramos cells (F) were treated in culture for 24 hours with the c-Myc inhibitor 10058-F4 (20μM) in the presence or absence of the broad spectrum caspase inhibitor QVD-OPH (10nM). Expression levels of Puma, Bcl-2, Bcl-6, and GAPDH (loading control) were examined by immunoblotting. (G) Tonsilar B cells (2 × 106 cells/mL) were activated with SAC (1/10 000) in the absence or presence of the c-Myc inhibitor 10058-F4 (20μM) for 24 hours: mRNA levels for Puma and GAPDH were analyzed for various cycles of PCR. Results are representative of 3 independent experiments.
