Cooperative enhancement of Trib1 and Evi1 for Hoxa9/Meis1-induced self-renewal and proliferation of myeloid progenitors. (A) Replating assays were performed using primary bone marrow cells infected with indicated retroviruses. The colony numbers in methylcellulose culture during 3 replatings were measured, and significant increase of colonies at the third replating is shown in Trib1/Hoxa9/Meis1 or Evi1/Hoxa9/Meis1 versus Hoxa9/Meis1. The means ± standard deviations from 3 independent experiments are shown. (B) Trib1 and Evi1 cooperatively promote myeloid-cell proliferation with Hoxa9 and Meis1. As in panel A, cumulative numbers of bone marrow cells in liquid culture medium were counted in the indicated duration. The means of the natural logarithm of cell numbers from 3 independent experiments are shown. Statistical analyses indicated that the difference of growth curves was significant between Trib1/Hoxa9/Meis1 and Hoxa9/Meis1 (P = .002) and between Evi1/Hoxa9/Meis1 and Hoxa9/Meis1 (P < .001). (C) Expression of epitope-tagged Meis1 (HA), Hoxa9 (myc), Evi1 (FLAG), and Trib1 (FLAG) in bone marrow cells was confirmed by Western blotting. β-tubulin was used as a loading control.