Skip to main content
. 2010 Aug 2;30(19):4575–4594. doi: 10.1128/MCB.01595-09

FIG. 9.

FIG. 9.

The increased levels of β-catenin protein and activity in Bcr/abl+ myeloid progenitor cells are Gas2 dependent. (A) The increase in β-catenin protein in Bcr/abl+ myeloid progenitor cells is reversed by Gas2 knockdown or the expression of DN-Gas2. WT murine myeloid progenitor cells were transduced with retroviral vectors expressing Bcr/abl (or the murine stem cell virus [MSCV] vector control) and either a Gas2-specific shRNA (or scrambled shRNA control) or DN-Gas2 (or the vector control). The lysate proteins from the transduced cells were analyzed by Western blot (WB) assay for β-catenin, Gas2, and tubulin (as a loading control). (B) Gas2 knockdown or the expression of DN-Gas2 reverses GM-CSF hypersensitivity in Bcr/abl+ murine myeloid progenitor cells. Murine myeloid progenitor cells from WT mice were cultured in GM-CSF, IL-3, and SCF and transduced with retroviral vectors as described above. Other cells were cotransduced with retroviral vectors to express Bcr/abl and a β-catenin-specific shRNA (or scrambled control). The cells were deprived of cytokines and treated with a dose titration of GM-CSF as indicated. Proliferation was determined by the incorporation of 3H-thymidine. *, **, or ***, statistically significant decrease in proliferation in Bcr/abl+ cells transduced with a DN-Gas2 vector versus the control MSCV vector, with a Gas2 shRNA vector versus the control, or with a β-catenin shRNA vector versus the control, respectively. CPM, counts per minute. (C) Gas2 knockdown or the expression of DN-Gas2 slightly decreases GM-CSF-induced proliferation in WT murine myeloid progenitor cells. Murine myeloid progenitor cells were transduced with retroviral vectors to express DN-Gas2 (or the empty vector control) or a Gas2-specific shRNA or a β-catenin-specific shRNA (or the scrambled shRNA control vectors). Proliferation in response to a dose titration of GM-CSF was determined as described above. *, **, or ***, statistically significant decrease in proliferation under the respective conditions. (D) The increase in β-catenin protein in Bcr/abl+ U937 cells is reversed by the overexpression of Gas2. Stable U937 transfectants were generated with a vector to express Bcr/abl or the empty vector control and a vector to express dominant-negative Gas2 (DN-Gas2) or a Gas2-specific shRNA (or the relevant control vectors). The cell lysates were analyzed by Western blot assay for the expression of β-catenin, ICSBP, Gas2, Bcr, or GAPDH (as a loading control). (E) The increased activity of a β-catenin-activated reporter construct in Bcr/abl+ U937 cells is reversed by Gas2 inhibition. U937 cells were cotransfected with the TOPflash reporter vector or the FOPflash control, a vector to express Bcr/abl or the empty vector, and a vector to express DN-Gas2 or a Gas2-specific shRNA (or the relevant control vectors). *, statistically significant increase in TOPflash reporter activity in cells expressing Bcr/abl (P < 0.0001, n = 4); ** or ***, statistically significant decrease in TOPflash reporter activity in transfectants with Bcr/abl and DN-Gas2 or Bcr/abl and shGas2, respectively, versus Bcr/abl alone (P < 0.0001, n = 4). The effect of these vectors on β-catenin protein expression is indicated by the results in the inset Western blot. Error bars indicate standard errors.