Figure 2

Anti-CD180 mediated phosphorylation of BTK and PI3K, expression of Mcl-1, Bcl-xL and apoptosis in AKT-S, p38MAPK-S and DS CLL cells. (A) Percentages of cells expressing p-BTK in AKT-S, p38MAPK-S and DS categories of CLL samples. Cells were incubated with anti-CD180 mAb for 20 min (CD180 mAb) or left unstimulated (Medium), washed, stained with anti-CD19 mAb, fixed, permeabilized and stained with anti-p-BTK mAb as described in the Materials and Methods and analyzed by flow cytometry. P values were calculated using paired t test. (B) Representative immunoblots show the levels of total PI3K, p-PI3K, Bcl-xL and Mcl-1 in AKT-S, p38MAPK-S and DS categories of CLL samples following stimulation with anti-CD180 mAb (CD180 mAb) or in unstimulated cultures (Medium) as described in the Materials and Methods. Bcl-2 was used as loading control. (C) Relative optical density, normalized to Bcl-2, for p-PI3K, Mcl-1 and Bcl-xL immunoblots, n = 6. (D) The percentages of DiOC₆^dim (apoptotic) cells in control B cells, AKT-S, p38MAPK-S and DS CLL samples measured following stimulation with anti-CD180 mAb for 24 h, compared with unstimulated cultures (Medium), as described in the Materials and Methods. The values are mean ± SD, p values were calculated using a nonparametrical U test. (E) Percentages of cells expressing p-AKT and p-p38MAPK in unstimulated (US) and stimulated with anti-CD180 mAb AKT-S and p38MAPK-S CLL samples untreated (white bars) or pretreated (gray bars) with specific inhibitors AKTi1/2 or SB2035804. P values between the treated and untreated with inhibitors cells were calculated using the paired t test.