Molecular mechanisms that determine responses of DLBCL to SYK inhibition or reduction. (A) Dose-dependent inhibition of BCR signaling in sensitive but not resistant DLBCL cell lines. Simultaneous phospho-flow analyses of p-PLCγ2 (left column), p-AKT (middle column), and p-ERK1/2 (right column) in DLBCL cells lines indicated on the right. Red traces indicate isotype control; green, cells stimulated with anti-BCR in the absence of PRT318; blue, stimulated cells treated with a 0.01μM concentration of inhibitor; and brown, stimulated cells treated with a 0.25μM concentration of drug. (B) Effects of SYK siRNA on PLCγ2 and AKT phosphorylation. LY1, LY7, LY18, and LY4 were transfected with SYK (blue traces) or control (green) siRNA pools. Phosphorylation was measured by the phospho-flow assays 48 hours after transfection. (C) Top panels show immunoblots of PLCγ2 or AKT protein in LY18 and LY4 cells transfected with PLCγ2 (+), AKT (+), or control siRNA (-) pools. GAPDH was blotted as a loading control. Bottom panels show simultaneous cell counting conducted at the indicated times after transfection. The fold increase in cell number indicates viable cell numbers relative to those at time 0. (D) Inhibition of BCR signaling in primary DLBCL lymphoma cells. Simultaneous phospho-flow analyses of p-PLCγ2 (left column), p-AKT (middle column), and p-ERK1/2 (right column) in primary DLBCL tumor cells. Color legends are as in panel A. Pt indicates patient.