Increased expansion of HSCs in Slug^−/− mice after 5-FU treatment. (A) A representation of flow cytometric analysis of Lin⁻Sca-1⁺ HSCs in Slug^+/+, Slug^+/−, and Slug^−/− BM 7 days after a single injection of 5-FU (300 mg/kg body weight). (B) The ratio of remaining Lin⁻Sca-1⁺ cells at 7 days after 5-FU treatment in 3 groups of mice (Slug^+/+ mice, n = 5; Slug^+/− mice, n = 4; Slug^−/−, n = 5). (C) Total numbers of surviving Lin⁻Sca-1⁺ cells 7 days after 5-FU treatment in Slug^+/+ (n = 5), Slug^+/− (n = 4), and Slug^−/− (n = 5) mice. (D) Analysis of apoptotic rate in primitive hematopoietic cell population in BM using an annexin V–based apoptosis assay. BM cells were harvested from Slug^−/− and Slug^+/+ mice at 0, 3, and 7 days after 5-FU treatment. Primitive hematopoietic cells were gated as Lin⁻Sca-1⁺c-Kit⁺ (for 0 day) or Lin⁻Sca-1⁺ (for 3 and 7 days after 5-FU treatment), and the apoptotic cells were identified by a fluorescence-labeled annexin V. Apoptotic primitive hematopoietic cells (annexin V⁺ Lin⁻Sca-1⁺c-Kit⁺ or annexin V⁺ Lin⁻Sca-1⁺) were analyzed by flow cytometry and represent a percentage. There is no significant difference in the percentage of annexin V⁺ Lin⁻Sca-1⁺c-Kit⁺ cells (0 day) and annexin V⁺ Lin⁻Sca-1⁺ cells (3 and 7 days after 5-FU treatment) from Slug^+/+ and Slug^−/− mice. The data shown are the mean of the triplicate. (E) Analysis of Lin⁻Sca-1⁺ cell population in S phase. Total BM cells were harvested from Slug^+/+ and Slug^−/− mice at 5 days after 5-FU treatment and then assayed for Edu incorporation (n = 3). The ratio of Edu⁺ cell in Lin⁻Sca-1⁺ cell population was determined by flow cytometry. All data are expressed as mean ± SD. *P < .05 and **P < .01 for Slug^−/− vs Slug^+/+.