STAT1 activation by Bryostatin 1 is mediated by IFNγ. (A) CLL cells were untreated (lane 1) or treated with 10 nM Bryostatin 1 (lane 4) for 3 hours. Conditioned medium from CLL cells stimulated with 10 nM Bryostatin 1 for 3 hours was incubated with untreated CLL cells for 15 minutes (lane 5). To test whether IFNγ was responsible for STAT1 activation, 0.5 μg/mL anti-IFNγ neutralizing antibody (αIFNγ) was incubated with conditioned medium for 20 minutes prior to addition to untreated CLL cells (lane 6). CLL cells were stimulated with 500 U/mL IFNγ for 15 minutes as a positive control (lane 2). To verify the neutralizing activity of the anti-IFNγ antibody, 50 U/mL IFNγ were incubated with 0.5 μg/mL anti-IFNγ antibody for 10 minutes prior to incubation with CLL cells for 15 minutes (lane 3). Whole cell lysates were prepared and analyzed for STAT1 tyrosine phosphorylation (Tyr-P-STAT1) by Western blotting. (B) RT-PCR for IFNγ was performed on mRNA from CLL cells left untreated for 1.5 hours or treated with 10 nM Bryostatin 1 for 1.5 to 3 hours. Amplification of GAPDH was performed as a control. (C) IFNγ ELISA was performed on culture supernatants collected from CLL cells untreated or pretreated with 100 μM AG490, 100 μM U0126, 500 μM Gö6976, or 10 μM Gö6850 for 1 hour prior to stimulation with 10 nM Bryostatin 1 for 3 hours. The relative IFNγ production is represented by normalizing the level of IFNγ produced under each condition to that of the Bryostatin 1–treated cells and is expressed as the mean percentage ± SEM from 4 patients.