Figure 1. Hoxa9 and Meis1 cooperation in leukemogenesis: leukemic cell engraftment is supported by MEIS1.

(A) Comparison of the proliferation and colony-forming activities of H9M1 cells expressing (+) or lacking (–) Meis1. MEIS1 expression in H9M1 and HΔM cells was confirmed by Western blotting; blots are representative of 2 experiments. Cumulative numbers of H9M1 cells in liquid culture were counted. Means of the natural logarithm of cell numbers from 3 independent experiments are shown (*P < 0.05, 2-tailed Student’s t test). Colony numbers per 1,000 H9M1 cells in methylcellulose culture were measured, and representative culture plates are shown (3 experiments). (B) Leukemia-free survival of sublethally irradiated animals transplanted with 1 × 10⁶ H9M1 cells are shown for H9M1 cells with (red line) or without (blue line) Meis1. (C) H9M1 cells in bone marrow 48 hours after transplantation were detected as an mKO-positive fraction by flow cytometry. A significant reduction of H9M1 cells in bone marrow was observed in animals transplanted with Meis1 KO cells. The number was restored by reintroduction of Meis1. Frequencies of mKO-positive cells in bone marrow are indicated as mean ± SEM of 3 independent experiments (**P < 0.01, 1-way ANOVA with Dunnett’s multiple comparison test). (D) Representative images of H9M1 cells in frozen bone marrow sections (3 experiments). DiO-stained H9M1 cells were detected, though they were absent in Meis1 KO mice, and were observed after Meis1 reintroduction into HΔM cells. Gr-1 is indicated by red fluoro-dye, and nuclei were counterstained with DAPI. Scale bar: 20 μm. (E) Engraftment activities were assessed by flow cytometry, which detected H9M1 or HΔM cells in bone marrow 2 weeks after transplantation as mKO-positive fractions. Data are representative of 3 independent experiments. (F) Coculture of H9M1 cells with OP9 cells. Cobblestone areas were established by H9M1 cells, but not by Meis1 KOs, and were restored by Meis1 reintroduction. Scale bar: 100 μm. Numbers of cobblestone areas (CFAs) are indicated as mean ± SEM of 3 independent experiments (**P < 0.01, 1-way ANOVA with Dunnett’s multiple comparison test).