Mef2c-deficient cells proliferate in vivo but show reduced homing and motility. (A) Percentage of BrdU incorporation after a 2-hour pulse in MLL/ENL-YFP-infected Mef2c^Δ/− MxCRE or Mef2c^+/+ MxCRE BM cells transplanted into irradiated mice. Each square represents an independent mouse receiving BM with the indicated phenotype. Cells were sorted for YFP expression (19%-34% of total BM) before staining for BrdU. (B) BM cells from Mef2c^Δ/− MxCRE or Mef2c^+/+ MxCRE mice (treated with pIpC) were labeled in vitro with CFSE and then injected via the tail vein into lethally irradiated syngeneic recipient mice. Shown is the relative mean proportion (± SE) of dye-positive cells from BM or spleen isolated 4 hours after transplantation from 4 mice from 2 independent experiments. An average of 68 (± 9.5) and 78 (± 32) CFSE⁺Mef2c^+/+ cells per 10⁵ total spleen or BM cells, respectively, were detected. (C) Visualization of MS-5 stroma cell cultures 24 hours after 10⁶ M/E Mef2c^+/+ or Mef2c^Δ/− cells were seeded onto monolayer. Cells transcending the monolayer are clearly seen as phase-dark cells (indicated by ↙). White translucent cells are above the stroma layer. The experiment was repeated 3 times with identical results. (D) Migration studies were compared between M/E cells in culture in which Mef2c was either deleted (by infection with CRE vector) or in which Mef2c was reintroduced with a Mef2c vector. Migration index was calculated 3 to 5 hours after cells from freshly infected and selected cultures were seeded into the upper well of a Transwell with SDF1α in the bottom chamber. Shown is the mean (± SE) of 2 independent experiments, each performed in duplicate. P values were calculated by the Student t test for each pair. (E) Migration index of M/E cells deficient for Mef2c that were either infected with an empty (pac) vector or a vector expressing the HDAC-biding mutant Mef2c^ASR. Migration index was calculated 3 to 5 hours after cells from freshly infected and selected cultures were seeded into the upper well of a Transwell with either PBS or SDF1α in the bottom chamber, as indicated. Shown is the mean (± SE) of 2 independent experiments, each performed in duplicate. P values were calculated by the Student t test for each pair. (F) Survival curves of mice receiving MLL/ENL-transduced Mef2^fl/fl BM, half of which received pIpC injections at the indicated times. (G) Verification of complete excision of the Mef2c floxed alleles after pIpC injection, PCR analysis was performed on DNA isolated from BM cells of diseased mice (3 from each cohort). Primers were designed to detect either the floxed (fl) or wild-type (wt) allele. Faint wt alleles can be detected in all animals resulting from residual host cells. Tail DNA from different genotypes was used as positive controls.