Figure 7.

MLL dominant negative mutants inhibit the colony-forming ability of wild-type myeloid progenitors. A, experimental procedure (see Materials and Methods). B, interaction of transduced MLL dominant negative mutants with endogenous menin in GFP-sorted BM cells. Equal amounts of cell extracts from GFP-positive–sorted BM cells were immunoprecipitated with M2 anti-FLAG monoclonal antibody and then immunoblotted with anti-menin polyclonal antibodies. A 5% aliquot of each cell lysate was analyzed by Western blot as control for endogenous levels of menin (Input). C, results of methylcellulose colony-forming assay summarized as a percentage of GFP-positive CFU obtained from 2 × 10⁴ transduced cells at 7 to 10 d from plating. Wild-type myeloid progenitor cells transduced with MLL dominant negative fragments with high menin-binding avidity show a reduced percentage of GFP-positive colonies compared with cells transduced with MLL fragments with weak or no interaction with menin (MLL2–35) or GFP alone. Bars, SD. D, morphology and density of GFP-positive colonies at 14 d from second methylcellulose plating. E, Wright-Giemsa–stained cytospins of cells obtained from colonies at the second round of plating in methylcellulose.