Figure 1
Figure 1. SHP2 is essential for constitutive growth of cells bearing oncogenic KITD814V. (A) The 32D cells were transduced with retrovirus bearing WT KIT or KITD814V as described in “Expression of WT and mutant KIT receptors in 32D cells and primary HSC/Ps.” Cells bearing WT KIT or KITD814V were starved of serum and growth factors for 6 hours followed by treatment with SHP2 inhibitor, II-B08 (20μM), for 1 hour, after which cells were lysed and equal amount of protein lysates were subjected to Western blot analysis using an anti–phospho-SHP2 (Y580) or total SHP2 antibody. Similar results were observed in 3 independent experiments. (B) The 32D cells bearing WT KIT or KITD814V were starved of serum and growth factors for 6 hours and subjected to proliferation assay in the presence or absence of indicated concentration of II-B08. Assay was performed in the presence of IL-3 (10 ng/mL) for cells bearing WT KIT and in the absence of growth factors for cells bearing oncogenic KITD814V. Bars represent the mean thymidine incorporation (CPM ± SD) from 1 of the 3 independent experiments performed in quadruplicate. *P < .005. (C) Primary BM-derived WT KIT or KITD814V-expressing cells from WT mice were starved for 6 hours and subjected to thymidine incorporation assay in the presence or absence of indicated concentrations of II-B08. Assays were performed in the presence of murine SCF (50 ng/mL) for cells bearing WT KIT and in the absence of growth factors for cells bearing oncogenic KITD814V. Bars represent the mean thymidine incorporation (CPM ± SD) from 1 of 3 independent experiments performed in quadruplicate. *P < .001. (D) Primary BM-derived cells expressing WT KIT or KITD814V from WT or SHP2−/− mice were starved and subjected to proliferation assay in the absence of growth factors by thymidine incorporation. Bars represent the mean thymidine incorporation (mean ± SD) from 1 of 3 independent experiments performed in quadruplicate. *P < .01, WT-KITD814V vs SHP2−/−-KITD814V. (E) Primary BM-derived cells expressing KITD814V from WT or SHP2−/− mice were starved and subjected to proliferation assay in the presence or absence of II-B08 (5μM) by thymidine incorporation. Bars represent the mean thymidine incorporation (mean ± SD) from 1 of 2 independent experiments performed in triplicate. *P < .05, WT-KITD814V-0μM vs WT-KITD814V-5μM.

SHP2 is essential for constitutive growth of cells bearing oncogenic KITD814V. (A) The 32D cells were transduced with retrovirus bearing WT KIT or KITD814V as described in “Expression of WT and mutant KIT receptors in 32D cells and primary HSC/Ps.” Cells bearing WT KIT or KITD814V were starved of serum and growth factors for 6 hours followed by treatment with SHP2 inhibitor, II-B08 (20μM), for 1 hour, after which cells were lysed and equal amount of protein lysates were subjected to Western blot analysis using an anti–phospho-SHP2 (Y580) or total SHP2 antibody. Similar results were observed in 3 independent experiments. (B) The 32D cells bearing WT KIT or KITD814V were starved of serum and growth factors for 6 hours and subjected to proliferation assay in the presence or absence of indicated concentration of II-B08. Assay was performed in the presence of IL-3 (10 ng/mL) for cells bearing WT KIT and in the absence of growth factors for cells bearing oncogenic KITD814V. Bars represent the mean thymidine incorporation (CPM ± SD) from 1 of the 3 independent experiments performed in quadruplicate. *P < .005. (C) Primary BM-derived WT KIT or KITD814V-expressing cells from WT mice were starved for 6 hours and subjected to thymidine incorporation assay in the presence or absence of indicated concentrations of II-B08. Assays were performed in the presence of murine SCF (50 ng/mL) for cells bearing WT KIT and in the absence of growth factors for cells bearing oncogenic KITD814V. Bars represent the mean thymidine incorporation (CPM ± SD) from 1 of 3 independent experiments performed in quadruplicate. *P < .001. (D) Primary BM-derived cells expressing WT KIT or KITD814V from WT or SHP2−/− mice were starved and subjected to proliferation assay in the absence of growth factors by thymidine incorporation. Bars represent the mean thymidine incorporation (mean ± SD) from 1 of 3 independent experiments performed in quadruplicate. *P < .01, WT-KITD814V vs SHP2−/−-KITD814V. (E) Primary BM-derived cells expressing KITD814V from WT or SHP2−/− mice were starved and subjected to proliferation assay in the presence or absence of II-B08 (5μM) by thymidine incorporation. Bars represent the mean thymidine incorporation (mean ± SD) from 1 of 2 independent experiments performed in triplicate. *P < .05, WT-KITD814V-0μM vs WT-KITD814V-5μM.

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