Loss of Cxcl12 leads to increased G₀-to-G₁ cell-cycle entry and cell proliferation of HSPC. (A) LSK cell proliferation was tested by BrdU incorporation for 3 and 14 days and assayed by flow cytometry; statistical data are presented in the right bar graph (mean ± SEM; **P < .005; n = 6 for 3 days and n = 5 for 14 days). (B) BM LSK cells were dual-stained with Hoechst 33342 and Pyronin Y to reveal the cell-cycle status. The percentage of LSK cells in G₀ phase (Pyronin Y^low, 2N DNA) is demonstrated on the right bar graph (mean ± SEM; *P < .05; n = 8 each). (C) Hoechst 33342 and Pyronin Y dual staining of 14-day culture of wild-type BM cells seeded on either Cxcl12-WT or Cxcl12-cKO stromal layer. Percentage of lineage⁻ gated cells in G₀ phase was analyzed and is shown in the right panel (mean ± SEM; ***P < .001; n = 3). (D) Quantitative RT-PCR assay for mRNA expression level of homeostatic regulator genes in Cxcl12-WT or Cxcl12-cKO stroma (mean ± SEM; *P < .05; n = 4). (E) ELISA test for SCF in stromal culture supernatant (mean ± SEM; ***P < .001; n = 6).