AG490 blocks Bryostatin 1–induced CLL differentiation. (A) CLL cells were untreated or pretreated with 100 μM AG490 for 1 hour and then treated with 10 nM Bryostatin 1 for 3 hours. Whole cell lysates were prepared and analyzed by Western blotting using antibodies specific for the tyrosine phosphorylated form of STAT1 (Tyr-P-STAT1). Blots were stripped and reprobed for total STAT1. Results are representative of experiments performed on 5 patients' cells. (B) AG490 inhibits Bryostatin 1–induced CD22 up-regulation. CLL cells were untreated or pretreated with 100 μM AG490 (AG) and then treated with 10 nM Bryostatin 1 for 24 hours. CD5/CD19⁺ cells were analyzed for CD22 expression by immunostaining and flow cytometric analysis. Values of CD22 expression were normalized to the level of CD22 expression in the Bryostatin 1–treated cells and are expressed as the mean percentage ± SEM from 3 patients. (C) IgM ELISA was performed on culture supernatants from CLL cells untreated or pretreated with 100 μM AG490 and then treated with 10 nM Bryostatin 1 or 500 U IFNγ for 48 hours. The values of IgM production were normalized to the level of IgM produced by the Bryostatin 1–treated cells and are the mean percentage ± SEM from 3 patients.