Stromal contact induced SYK phosphorylation and SYK-dependent Akt phosphorylation and F-actin expression. (A) Coculture of CLL samples with the stromal cell line M2-10B4 (n = 6; P < .01), with primary bone marrow–derived stromal cells (n = 8; P < .01) or with the human stromal cell line HS-5 (n = 5; P = .06) resulted in an increase of pY352 SYK, determined by the mean fluorescent intensity (MFI). (B) Parallel stimulation of 9 CLL samples from pretreated patients and 10 samples from patients who have not been treated revealed differential amounts of SYK phosphorylation induced by stroma but not IgM stimulation in these subgroups with higher induction in the pretreated subset. n.s. indicates not significant. (C) Immunoprecipitation of SYK with subsequent immunoblotting for pY revealed SYK phosphorylation induced by 15 minutes of murine (top) or human (bottom) stromal cell contact shown for 3 representative CLL samples. For sample 225, only 5 μg protein lysate was used for the IP. (D) Bar diagram represents mean values ± SEM of MFI for phospho-Akt-stained primary CLL samples (n = 8) with and without M2-10B4 (left panel) or primary (right panel) stromal contact, in the presence and absence of SYK inhibitor R406. Displayed is a representative immunoblot of pAkt after 15 minutes of stromal cell coculture in the presence and absence of R406. (E) Murine and human stromal coculture induced F-actin formation in control-treated cells (P < .01); this was markedly reduced in R406 pretreated cells (P < .01) as determined by phalloidin-Alexa488 staining and flow cytometric analysis (n = 8).