Fig. 5: TG02 blocked BCR signaling in the CLL cells.

A CLL cells were cultured in RPMI media supplemented with 10% autologous plasma, either alone or at the presence of anti-IgM. The cells were incubated with increasing concentrations of TG02 and viabilities were analyzed by Annexin V/PI double staining at the end of the 24 h incubation. Data represents mean ± SE of four CLL samples. B TG02 blocked BCR signaling-mediated activation of NF-κB. CLL cells were incubated with TG02 for 1 h before stimulating with anti-IgM for 2 h, and NF-κB p65 activation and inhibition were measured by chemiluminescence and presented as the percentage of control (mean ± SEM) of three samples. Black bars: with anti-IgM stimulation; white bars: no stimulation. WT Oligo and Mut Oligo represent the wild-type and mutated p65 consensus binding oligonucleotides that were used to confirm the specificity of the analysis. C The phosphorylation status of kinases in the BCR signaling pathway was analyzed by immunoblotting. GAPDH was used as loading control. CLL cells were incubated with TG02 or Lcki for 1 h before stimulating with anti-IgM for 1 h and 24 h. A representative immunoblot of three experiments is shown. D Inhibition of BCR signaling by TG02 was mediated by the inhibition of Lck and JAK2. CLL cells were incubated with TG02, Lcki, SNS-032, Sunitinib, or TG-101348 for 1 h before stimulating with anti-IgM for 1 h. The phosphorylation of Akt and ERK was evaluated by immunoblotting. A representative immunoblot of three experiments is shown.