Figure 2
Figure 2. Treatment with GO-203 inhibits FLT3 activation and signaling. (A) Lysates from BaF3 cells grown in the presence of interleukin-3, BaF3/FLT3-ITD cells, and MOLM-14 cells were immunoblotted with the indicated antibodies. BaF3 and BaF3/FLT3-ITD express MUC1-C as the 17-kDa unglycosylated (un-gly) form and MOLM-14 cells express the 20- to 25-kDa glycosylated (gly) form. (B) BaF3/FLT3-ITD cells were left untreated (control) or treated with 5 μM GO-203 for 0.5 and 2 hours. Lysates were subjected to immunoblotting with anti-p-FLT3 (upper panel), anti-FLT3 (middle panel), and anti-β-actin (lower panel). (C) MOLM-14 cells were left untreated (control) or treated with 5 μM GO-203 for 30 minutes. Total cell lysates were then subjected to immunoprecipitation with anti-FLT3 antibody. The immunoprecipitates were then analyzed by immunoblotting with anti-P-Tyr (upper panel) or anti-FLT3 (middle panel). As control, total cell lysates were also analyzed by immunoblotting with anti-β-actin (lower panel). (D) Total cell lysates from MOLM-14 cells stably expressing a CshRNA or a MUC1shRNA were analyzed by immunoblotting with the indicated antibodies. (E) MOLM-14/CshRNA (diamonds) and MOLM-14/MUC1shRNA (squares) were seeded at 0.5 × 105 cells/mL. Cell number (mean ± SD of 3 replicates) was determined on the indicated days. (F) MOLM-14 cells were left untreated (control) or treated with 5 μM GO-203 for 60 minutes. Lysates were subjected to immunoblotting with anti-p-AKT (upper panel), anti-AKT (middle panel), and anti-β-actin (lower panel). (G) MOLM-14 cells were left untreated (control) or treated with 5 μM GO-203 for 30 and 60 minutes. Lysates were subjected to immunoblotting with anti-p-ERK antibody (upper panel), anti-ERK antibody (middle panel), and anti-β-actin antibody (lower panel). (H) Primary AML cells were treated with 4 μM GO-203 for 4 hours and then incubated with a combination of anti-human PE-Cy7 CD13 and CD33. For staining, cells were incubated with an anti-human APC-conjugated FLT3 (total FLT3) or APC isotype control antibody. After washing and fixing, the cells were incubated with the combination of anti-human APC-conjugated p-ERK1/2, nonconjugated p-FLT3 (Y591), or the corresponding combination of isotype control antibodies. Following incubation with an Alexa Fluor 488 goat anti-rabbit antibody, samples were analyzed using a FACSCanto II analyzer. A representative flow profile of AML cells from patient 2 is shown in the upper panels. The results are also expressed as the ratio (treated/control; T/C) of fluorescence found for blasts from patients 1-3 (lower panels). (I) BaF3/FLT3-ITD cells were left untreated (control) or treated with 2 μM GO-203 for 2 hours. Total cell lysates were subjected to immunoblotting with anti-p-STAT5 (upper panel), anti-STAT5 (middle panel), and anti-β-actin (lower panel).

Treatment with GO-203 inhibits FLT3 activation and signaling. (A) Lysates from BaF3 cells grown in the presence of interleukin-3, BaF3/FLT3-ITD cells, and MOLM-14 cells were immunoblotted with the indicated antibodies. BaF3 and BaF3/FLT3-ITD express MUC1-C as the 17-kDa unglycosylated (un-gly) form and MOLM-14 cells express the 20- to 25-kDa glycosylated (gly) form. (B) BaF3/FLT3-ITD cells were left untreated (control) or treated with 5 μM GO-203 for 0.5 and 2 hours. Lysates were subjected to immunoblotting with anti-p-FLT3 (upper panel), anti-FLT3 (middle panel), and anti-β-actin (lower panel). (C) MOLM-14 cells were left untreated (control) or treated with 5 μM GO-203 for 30 minutes. Total cell lysates were then subjected to immunoprecipitation with anti-FLT3 antibody. The immunoprecipitates were then analyzed by immunoblotting with anti-P-Tyr (upper panel) or anti-FLT3 (middle panel). As control, total cell lysates were also analyzed by immunoblotting with anti-β-actin (lower panel). (D) Total cell lysates from MOLM-14 cells stably expressing a CshRNA or a MUC1shRNA were analyzed by immunoblotting with the indicated antibodies. (E) MOLM-14/CshRNA (diamonds) and MOLM-14/MUC1shRNA (squares) were seeded at 0.5 × 105 cells/mL. Cell number (mean ± SD of 3 replicates) was determined on the indicated days. (F) MOLM-14 cells were left untreated (control) or treated with 5 μM GO-203 for 60 minutes. Lysates were subjected to immunoblotting with anti-p-AKT (upper panel), anti-AKT (middle panel), and anti-β-actin (lower panel). (G) MOLM-14 cells were left untreated (control) or treated with 5 μM GO-203 for 30 and 60 minutes. Lysates were subjected to immunoblotting with anti-p-ERK antibody (upper panel), anti-ERK antibody (middle panel), and anti-β-actin antibody (lower panel). (H) Primary AML cells were treated with 4 μM GO-203 for 4 hours and then incubated with a combination of anti-human PE-Cy7 CD13 and CD33. For staining, cells were incubated with an anti-human APC-conjugated FLT3 (total FLT3) or APC isotype control antibody. After washing and fixing, the cells were incubated with the combination of anti-human APC-conjugated p-ERK1/2, nonconjugated p-FLT3 (Y591), or the corresponding combination of isotype control antibodies. Following incubation with an Alexa Fluor 488 goat anti-rabbit antibody, samples were analyzed using a FACSCanto II analyzer. A representative flow profile of AML cells from patient 2 is shown in the upper panels. The results are also expressed as the ratio (treated/control; T/C) of fluorescence found for blasts from patients 1-3 (lower panels). (I) BaF3/FLT3-ITD cells were left untreated (control) or treated with 2 μM GO-203 for 2 hours. Total cell lysates were subjected to immunoblotting with anti-p-STAT5 (upper panel), anti-STAT5 (middle panel), and anti-β-actin (lower panel).

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