Figure 1

Blk(Y501F) drives resistance to apoptosis and cytokine-independent proliferation. (a) Ba/F3 Blk-wt and Ba/F3 Blk(Y501F), as well as non-transfected Ba/F3 cells (NT), were cultured in the absence of IL-3. Graph to the left depicts the percentage of viable cells at different time points, where each data point represents the mean of a duplicate-sample count. The graph to the right depicts the total number living cells. The data are representative of two independent experiments. (b) Ba/F3 NT, Ba/F3 Blk-wt and Ba/F3 Blk(Y501F) were cultured with or without IL-3 for 6 days before apoptosis was determined by 7-aminoactinomycin D (7-AAD) staining using flow cytometry. Percentages in upper left corner of individual graphs indicate mean percentages of apoptotic cells from three independent experiments, of which the data shown are representative. (c) Western blot (WB) analysis using antibodies specific for Blk-activating phosphotyrosine-388, and antibodies recognizing total Blk, of NT, Blk-wt, and Blk(Y501F) (pre-selection) Ba/F3 cells after 16 h of IL-3 deprivation, as well as of IL-3-independent Ba/F3 Blk(Y501F) cells post-selection established in the experiment depicted in (a). GAPDH was used as loading control. (d) WB analysis using antibodies targeting the Blk-deactivating phoshotyrosine-501 in Ba/F3 NT cells and stable Ba/F3 transfectants. GAPDH was used as loading control. (e) Ba/F3 NT and IL-3-independent selected Ba/F3 Blk(Y501F) cells were treated with different concentrations of either LckI, p38 inhibitor (p38I) or vehicle (DMSO (dimethyl sulfoxide)) for 48 h before cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data points represent mean percentage viability of LckI- and p38I-treated cells, relative to vehicle-treated controls, from median values of three independent experiments performed in triplicates, with error bars representing ±s.e.m. Only the IL-3-dependent Ba/F3 NT cells were grown in IL-3-supplemented media. (f) IL-3-independent selected Ba/F3 Blk(Y501F) cells were treated with 10 μM of dasatinib or vehicle (DMSO) for 1 h before the cells were lysed and WB analysis using anti-pY³⁸⁸-Blk and anti-Blk antibodies was performed. STAT5B and GAPDH served as loading controls. (g) Ba/F3 NT and IL-3-independent selected Ba/F3 Blk(Y501F) cells were treated with different concentrations of either dasatinib, LckI or vehicle (DMSO) for 48 h before cell viability was determined by MTT assay. Data points represent mean percentage viability of dasatinib- and LckI-treated cells, relative to vehicle. Only the IL-3-dependent Ba/F3 NT cells were grown in IL-3-supplemented media.