Rbm15-Mkl1 Interacts with the Setd1b Histone H3-Lys4 Methyltransferase via a SPOC Domain That Is Required for Cytokine-Independent Proliferation
Figure 8
Rbm15-Mkl1 enhances the proliferation of murine 6133 megakaryoblastic leukemic cells in a SPOC domain dependent manner.
A. Diagram of pCL6IEGwo lentiviral expression constructs. GFP is expressed from an internal ribosome entry site (IRES). B. Murine 6133 megakaryoblastic leukemic cells were transduced with lentivirus encoding Rbm15, Rbm15 (K795A), Rbm15-Mkl1, Rbm15-Mkl1 (K795A), or with empty vector. After four days of transduction, GFP-positive cells were sorted and cultured in medium containing 10 ng/ml SCF. Whole cell extracts were prepared and analyzed by Western blotting using the indicated antisera. Arrows indicate the position of Rbm15, Mkl1, and Rbm15-Mkl1. C. GFP-positive 6133 megakaryoblastic leukemic cells were cultured in medium containing SCF and cells were enumerated every two or three days after staining with trypan blue. The error bars indicate the mean standard deviation from three experiments, and asterisks indicate p values compared to vector control (*, p = 0.008; **, p = 0.001, ***, p = 0.0001). D. Analysis of apoptosis in transduced 6133 megakaryoblastic leukemic cells. GFP-positive 6133 leukemic cells were analyzed for apoptosis using APC-annexin V and 7-ADD staining and flow cytometry. UR, upper right panel corresponding to dead cells; LL, lower left panel corresponding to healthy cells; LR, lower right panel corresponding to apoptotic cells. Numeric values represent a summary of data from three experiments. Asterisks indicate p values compared to vector control (p<0.05).