Figure 3

Pharmacological inhibition of Trk receptors sensitises the effect of rituximab in ABC and GCB cell lines. GCB- (SUDHL4) and ABC (OCI-LY10)-like DLBCL cells were exposed to rituximab (RTX, 1 and 20 μg ml⁻¹) with or without (C, controls) pharmacological inhibitor of Trk receptors, K252a (350 nM). (A) Inhibition of Trk signalling by K252a was confirmed in both cell lines by western blot analysis of TrkA, TrkB and TrkC receptor phosphorylation. (B) Viability of cells was evaluated during 48 h and 72 h using the XTT test and data are expressed as means±s.d. of relative cell viability (ratio) related to control obtained from four independent experiments. **P<0.01 and ***P<0.001 when compared with DMSO controls (Student’s t-test). (C) Apoptosis induced by rituximab and/or K252a was analysed by flow cytometry using the Annexin-V-FITC/PI dual staining. Ratio of early and late apoptotic (lower right quadrant) and necrotic (upper right quadrant) cells obtained after 36 h cell culture are expressed as cell percentages. Example of flow cytometric analysis representative of three experiments is done. (D) Rituximab (R) or/and K252a (K) induced apoptosis was also evaluated by western blot analysis of the poly (ADP-ribose) polymerase (PARP) cleavage after 48 h exposure in cell lysates. Data are representative of three independent experiments. Actin expression is done as loading reference. Abbreviations: FL, full-length (FL); CL, cleaved PARP.