Skp2 expression reflects the cell-cycle status of hematopoietic cells during differentiation. (A) BM was harvested from C57BL/6J mice 24 hours after injection with BrdU and labeled in conjunction with the surface markers necessary to identify the indicated cell subsets. BrdU incorporation and DNA content (7-AAD) were measured in gated LSK, LK and Gr1⁺Mac1⁺ populations. Bars represent average of cells positive for BrdU. n = 4-6 of 3 independent experiments. *P < .05 vs LSK. (B) Fold change in Skp2 mRNA expression by quantitative RT-PCR in sorted LSK, LT-HSC, ST-HSC, LK, CMP, GMP, MEP, and Gr1⁺Mac1⁺ populations. n = 2-4 samples (each sample a pool of 5-6 mice) in 2 independent experiments. *P < .05 vs LSK #P < .05 vs LK. (C) Purified Lin⁻ cells were cultured in the presence of SCF and IL-3 for 0, 2, or 4 days and analyzed for S-phase by BrdU incorporation (3-hour pulse) and DNA content (7-AAD); values in the bar graph represent average of percentage of cells in S-phase in 2 experiments. (D) Relative expression of Skp2 by q-RT-PCR in Lin⁻ cells harvested at day 0, 2, and 4 of culture. (E) Kinetics of myeloid differentiation measured as expression of Gr1 and Mac1 by immunophenotypic analysis. Values are average of cells expressing Gr1⁺Mac1⁺ in the total population. In panels D and E, n = 4-6 samples in 3 independent experiments. Data are expressed as mean ± SEM *P < .05 vs day 0. ^#P < .05 vs day 2.