Figure 3
Figure 3. The effect of SRC inhibition on FLT3 mutant–expressing cells. (A) Cells (32D) stably expressing FLT3-ITD or FLT3-TKD were cultured in the presence of the inhibitor PD166-326 at the indicated concentrations with or without IL-3. The proliferation was measured after 48 hours using an MTT-based assay. Data are presented as the percentage of DMSO-treated cells and represent values ± SD of triplicates. One representative of at least 3 independent experiments is shown. Statistical analysis was conducted. ***P < .001; **P < .01; *P < .05; and n.s. (no significance), P > .05. (B) Cells (32D FLT3-ITD) were treated with PD166-326 in serum-free and cytokine-free medium for 4 hours. The lysates were analyzed for STAT5 activation by immunoblotting. Immunoprecipitation of FLT3-ITD, followed by immunoblotting with pY and FLT3 Abs, was performed. Phosphorylation of SRC was analyzed by immunoprecipitation, followed by immunoblotting with anti-pY Ab. SRC expression was verified by anti-SRC immunoblotting. Activation of STAT5 was investigated by immunoblotting using pSTAT5 Ab. To confirm equal expression of FLT3 and equal loading, the membrane was reprobed with FLT3 and STAT5 Abs. (C-D) Cells (32D) stably expressing FLT3-ITD or FLT3-TKD were incubated for 48 hours with PD166-326 and PKC412 as indicated. The proliferative activity of the cells was determined by MTT assay. Data are presented as the percentage of DMSO-treated cells and represent values ± SD of triplicates. One representative of at least 3 independent experiments is shown. The median combinatory index for FLT3-ITD–expressing cells for effective dose 50 and 75 was determined as 0.34 using CalcuSyn Version II software (Biosoft) and indicated synergism. (E) Cells (32D) as described in panel A were starved for 6 hours in serum-free medium and treated with 125nM PD166-326 and 5nM PKC412 or PKC412 alone, and then the cells were lysed and subject to immunoblot analysis. The activation of STAT5 was determined using pSTAT5 Ab. To ensure equal loading and expression, the membranes were stripped and reprobed with FLT3 and ACTIN Abs.

The effect of SRC inhibition on FLT3 mutant–expressing cells. (A) Cells (32D) stably expressing FLT3-ITD or FLT3-TKD were cultured in the presence of the inhibitor PD166-326 at the indicated concentrations with or without IL-3. The proliferation was measured after 48 hours using an MTT-based assay. Data are presented as the percentage of DMSO-treated cells and represent values ± SD of triplicates. One representative of at least 3 independent experiments is shown. Statistical analysis was conducted. ***P < .001; **P < .01; *P < .05; and n.s. (no significance), P > .05. (B) Cells (32D FLT3-ITD) were treated with PD166-326 in serum-free and cytokine-free medium for 4 hours. The lysates were analyzed for STAT5 activation by immunoblotting. Immunoprecipitation of FLT3-ITD, followed by immunoblotting with pY and FLT3 Abs, was performed. Phosphorylation of SRC was analyzed by immunoprecipitation, followed by immunoblotting with anti-pY Ab. SRC expression was verified by anti-SRC immunoblotting. Activation of STAT5 was investigated by immunoblotting using pSTAT5 Ab. To confirm equal expression of FLT3 and equal loading, the membrane was reprobed with FLT3 and STAT5 Abs. (C-D) Cells (32D) stably expressing FLT3-ITD or FLT3-TKD were incubated for 48 hours with PD166-326 and PKC412 as indicated. The proliferative activity of the cells was determined by MTT assay. Data are presented as the percentage of DMSO-treated cells and represent values ± SD of triplicates. One representative of at least 3 independent experiments is shown. The median combinatory index for FLT3-ITD–expressing cells for effective dose 50 and 75 was determined as 0.34 using CalcuSyn Version II software (Biosoft) and indicated synergism. (E) Cells (32D) as described in panel A were starved for 6 hours in serum-free medium and treated with 125nM PD166-326 and 5nM PKC412 or PKC412 alone, and then the cells were lysed and subject to immunoblot analysis. The activation of STAT5 was determined using pSTAT5 Ab. To ensure equal loading and expression, the membranes were stripped and reprobed with FLT3 and ACTIN Abs.

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