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

FIG. 8.

FIG. 8.

The increased levels of β-catenin protein and activity in myeloid progenitor cells with ICSBP knockdown are Gas2 dependent. (A) The expression of DN-Gas2 or a Gas2-specific shRNA decreases β-catenin protein expression in ICSBP−/− myeloid progenitor cells. Bone marrow myeloid progenitors were isolated from WT and ICSBP−/− mice and cultured in GM-CSF, IL-3, and SCF. Cells were transduced with a vector to express dominant-negative Gas2 (DN-Gas2) or empty vector control. Some ICSBP−/− myeloid progenitors were transduced with a vector to express a Gas2-specific shRNA or scrambled control. The lysate proteins from the transduced cells were analyzed by Western blot assay for β-catenin and Gas2 protein expression. The blots were also probed with an antibody to tubulin as a loading control. (B) The expression of DN-Gas2 in WT or ICSBP−/− myeloid progenitor cells decreases the expression of β-catenin target genes. WT or ICSBP−/− myeloid progenitor cells were transduced with a vector to express DN-Gas2 or a Gas2-specific shRNA (or relevant controls). Total cellular RNA was isolated from the transduced myeloid progenitor cells and analyzed by real-time PCR for the expression of β-catenin or β-catenin target genes. The results were normalized to those for 18S RNA. *, #, or & ***, statistically significant decrease in the expression of c-myc, cyclin D1, or survivin, respectively, in ICSBP−/− cells with DN-Gas2 in comparison to their expression with the control vector (P < 0.001, n = 3); **, ##, or &&, statistically significant decrease in mRNA expression of c-myc, cyclin D1, or survivin, respectively, in cells transduced with shGas2 vector. (C) Gas2 knockdown or the expression of DN-Gas2 reverses GM-CSF hypersensitivity in ICSBP−/− myeloid progenitor cells. Murine myeloid progenitor cells from WT or ICSBP−/− mice were cultured in GM-CSF, IL-3, and SCF. Cells were transduced with a vector to express a Gas2-specific shRNA, a β-catenin-specific shRNA, dominant-negative Gas2 (DN-Gas2), or the relevant control vectors. WT cells were also transduced with empty murine stem cell virus (MSCV) vector. The cells were cytokine deprived 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 ICSBP−/− cells transduced with vector to express DN-Gas2 versus control MSCV vector or Gas2 shRNA or β-catenin shRNA versus control, respectively. CPM, counts per minute. (D) The expression of dominant-negative Gas2 reverses the increase in β-catenin protein in U937 cells with ICSBP knockdown. Stable U937 transfectants were generated with a vector to express an ICSBP-specific shRNA or scrambled control shRNA 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, Gas2, ICSBP, or GAPDH (as a loading control). (E) The increased activity of a β-catenin-binding reporter construct in U937 cells with ICSBP knockdown is reversed by dominant-negative Gas2, and its decreased activity in cells with ICSBP-overexpression is reversed by the overexpression of Gas2. U937 cells were cotransfected with the TOPflash reporter vector or FOPflash control, a vector to express an ICSBP-specific shRNA or scrambled shRNA control vector, and a vector to express dominant-negative Gas2 (DN-Gas2) or a Gas2-specific shRNA or the relevant control vectors. Other cells were cotransfected with the reporter vectors and vectors to overexpress ICSBP or the empty vector control and a vector to express Gas2 or the empty vector control. A dose titration of DN-Gas2, shGas2, and Gas2 expression vectors was performed. *, statistically significant increase in reporter activity in cells with an ICSBP-specific shRNA; ** or ***, statistically significant decrease in TOPflash reporter activity in transfectants with DN-Gas2 or Gas2-specific shRNA, respectively (P < 0.0001, n = 4); # and ##, statistically significant decrease in TOPflash reporter activity in transfectants with ICSBP and increase in transfectants with ICSBP plus Gas2, respectively, in comparison to ICSBP alone (P < 0.01, n = 4). The reporter activity in transfectants with shICSBP and either DN-Gas2 or shGas2 is not significantly different than that in the control, as indicated. Error bars indicate standard errors.