GABPα is required for HSC differentiation and survival. (A) Peripheral blood analysis. Blood was collected from Mx1Cre-GABPαFL/+ and Mx1Cre-GABPαFL/− mice on indicated days after pIpC treatments and analyzed on Sysmex XT 2000iv automatic hematology analyzer. Numbers of platelets and white blood cells (WBCs) are shown, with horizontal bars denoting mean values in each group. Data are pooled results from 2-3 independent experiments with 5-13 animals analyzed. (B) Colony formation assays using methylcellulose. Total BM cells from Mx1Cre-GABPαFL/+ and Mx1Cre- GABPαFL/− mice were isolated 4 days after the last pIpC injection. A total of 2 × 104 BM cells were mixed and plated with M3434 methylcellulose-based media. Colonies of each type were counted after 10-day culture. GEMM indicates granulocyte, erythroid, macrophage, megakaryocyte; GM, granulocyte macrophage; G, granulocyte; M, macrophage; BFU-E, burst-forming unit-erythroid. Data are representative from 2 independent experiments with 8 animals analyzed. The colony numbers are averages of triplicate measurements of each individual mouse. (C) Ablation of GABPα diminishes CFU-Spleen12 colonies. Mx1Cre-GABPαFL/+ and Mx1Cre-GABPαFL/− mice were treated with pIpC, and on day 9 after last injection, 1 × 105 BM cells were injected into 900 rad–irradiated hosts. After another 12 days, spleens from the hosts were harvested and macro-colonies were counted. Shown are representative from 2 independent experiments with similar results (n = 4 in each experiment). (D) Detection of apoptotic cells in LSK and myeloid progenitor subsets. Total BM cells were isolated from Mx1Cre-GABPαFL/+ and Mx1Cre-GABPαFL/− mice 2-4 days after pIpC treatment, lineage-depleted, and surface-stained with c-Kit and Sca-1, followed by staining with annexin V and 7-AAD. The percentage of annexin V–7-AAD+ cells in each subset is shown. (E) Enhanced cell death of GABPα-deleted LSKs and myeloid progenitors. Data were pooled results from 2 independent experiments with 6 mice of each genotype analyzed.
