CUX1 regulates HSPC numbers, proliferation, and lineage specification. Total BM cells isolated from dox-naïve Cux1^mid, Cux1^low, or littermate Ren mice were mixed with M3434 methylcellulose medium (25 000 cells per well) and dox for CFU assays. At each passage (P1-P4), cells were collected and 25 000 cells were replated, with 7 to 11 days between each passage. (A) Total colony numbers and (B) cell counts were quantified at the end of each passage (n = 3). (C) Specific colonies (CFU-GM, BFU-E, and CFU granulocyte, erythrocyte, monocyte, and megakaryocyte [CFU-GEMM]) were identified by morphology days 10 through 12 in a separate CFU assay. (D) BM cells were collected from Cux1^mid, Cux1^low, and Ren littermate controls after 3 days of dox. Lin⁻ cells were cultured in StemSpan SFEM medium (Stemcell Technologies) supplemented with 100 ng/mL of murine stem cell factor, throbopoietin, and Flt3 and dox for 3 days before flow analysis. Representative flow cytometric analysis of HSPC cell-cycle analysis is shown. (E) The bar graph represents the mean of 3 biological replicates ± SEM. All data were analyzed with Student t test, *P ≤ .05, **P ≤ .01, ***P ≤ .001.