Fig 2.

Effects of STAT3 mutations on haematopoietic precursors. (A) Representative dot plots of flow cytometric analysis of myeloid (Gr-1⁺/Mac-1⁺), erythroid (Ter119⁺/CD71⁺), megakaryocytic (CD61⁺/CD41⁺), B-cell (B200⁺) and T-cell (Thy-1⁺) precursors in the bone marrow (BM) and spleens of transplanted animals expressing STAT3^WT, STAT3^D661V and STAT3^Y640F at 16-20 weeks after bone marrow transplantation (BMT) are shown. (B) Percentages of myeloid (Gr-1⁺/Mac-1⁺), erythroid (Ter119⁺/CD71⁺), megakaryocytic (CD61⁺/CD41⁺), B-cell (B200⁺) and T-cell (Thy-1⁺) precursors in the BM and spleens (SPL) of transplanted animals expressing STAT3^WT (n=4), STAT3^D661V (n=4) and STAT3^Y640F (n=4) are shown in bar graphs as mean ± SEM. (C) Flow cytometric analysis of CD3⁺CD8⁺ cells in the peripheral blood (PB), BM and spleens of transplanted animals expressing STAT3^WT, STAT3^D661V and STAT3^Y640F at 16-20 weeks after BMT. (D) Percentages of CD3⁻ natural killer cells (CD3⁻NK1.1⁺) in the peripheral blood (PB), BM and spleens of transplanted animals expressing STAT3^WT, STAT3^D661V and STAT3^Y640F at 16-20 weeks after BMT are shown in histograms as mean ± SEM (n=4). (E) Flow cytometric analysis of GFP expression in the BM at 16-20 weeks after BMT. Data are shown in histograms as mean ± SEM (n=4; * indicates p<0.05 by Student's t-test). Note that significantly increased percentages of GFP⁺ cells in the BM of transplanted animals expressing STAT3^D661V or STAT3^Y640F compared with transplanted animals expressing STAT3^WT. (F) Immunoblot analysis on total BM extracts shows constitutive phosphorylation of STAT3 in the BM of transplanted animals expressing STAT3^D661V and STAT3^Y640F mutants, suggesting that the STAT3 mutants were efficiently expressed and constitutively activated in the BM of the transplanted animals. β-actin was used as a loading control.