ME^m1/m1 mice lack Cdkn1c expression in HSCs. (A) BrdU incorporation. LSKs were isolated from WT and ME^m1/m1 mice after 3 days of labeling; cells were harvested, and cytospins were stained for BrdU and counted. Ten independent analyses were performed (P = .0014 by χ² test). Error bars indicate SD. (B) Ki67 staining on glass slides. LSKs were harvested from WT and ME^m1/m1 BM, cytospun onto glass slides, and stained with antibody against Ki67. Data are based on 3 independent analyses; error bars indicate SD. (C) Ki67 staining by flow cytometry. Whole BM from WT and ME^m1/m1 mice was stained for the lineage markers c-Kit, Sca-1, and Ki67. Data are the percentage of Ki67-positive cells in the LSK population. Shown are 2 independent experiments. (D) Recovery from myeloablation by 5-FU. Cohorts of 15 ME^m1/m1 and 15 WT adult mice were given intraperitoneal injections of 5-FU on day 0, and monitored thereafter. Cumulative survival is charted against time in days. (E) Analysis of Cdkn1c transcript levels in WT and ME^m1/m1 LSKs by RNA-Seq; shown are RNA levels (in reads per million mapped reads) in 2 preparations for each genotype, with each preparation consisting of pooled LSKs from 7 mice. (F) Fraction of cells that immunopositive for the cell-cycle markers and regulators indicated, as assessed by diaminobenzidine development of HRP-conjugated avidin/biotinylated antibodies applied to cytospin smears of isolated LSKs from the genotypes indicated after transduction with the viruses indicated: V indicates empty vector; M, MDS1; E, EVI1; and ME, MDS1-EVI1. Error bars indicate SD. *P < .001 by Student t test.