Spontaneous and constitutive mobilization of HSCs in Sclpfn1 mice. (A) Sclpfn1 BM HSCs are less quiescent than control HSCs. (Left) LT-HSCs (as Lin⁻Sca-1⁺Kit⁺Flk2⁻CD34⁻ cells) from a WT mouse, stained with Hoechst 33342 and pyronin Y, were analyzed for cell cycle stage. (Right) The percentages of G0 cells in control and Sclpfn1 mice (n = 3-4) at days 4 and 8 after tamoxifen treatment are shown (*P < .05). (B) BrdU incorporation indicates a decreased cycling in HSCs isolated from control mice compared with Sclpfn1 mice (n = 3; *P < .05). (C) Relative frequency of LT-HSCs in peripheral blood (PB) was analyzed over time in control and Sclpfn1 mice after tamoxifen treatment (n = 3-7; *P < .05). (D) The migrations of LT-HSCs (as Lin⁻Sca-1⁺Kit⁺Flk2⁻CD34⁻ cells) isolated from control and Sclpfn1 mice 8 days after tamoxifen treatment were compared in a transwell experiment (n = 3; *P < .05). (E) Control and Sclpfn1 HSCs home equivalently to recipient BM. BM from control or Sclpfn1 mice (n = 5) was labeled with 5- and 6-carboxyfluorescein succinimidyl ester (CFSE), and 1 × 10⁷ cells were transplanted into lethally irradiated recipients. After 16 hours, the total percentage of CFSE⁺ cells in the BM, spleen, and liver and LT-HSCs (CFSE⁺Lin⁻Sca-1⁺Kit⁺ Flk2⁻CD34⁻ cells) in BM were determined by flow cytometry. (F) Lysates of WT mouse BM cells were coimmunoprecipitated with anti-Gα13 antibody or control rabbit IgG, and precipitation was then determined by western blotting using anti-Gα13 and anti-pfn1 antibodies (G-H) Rescue of Sclpfn1 LT-HSCs by EGR1. Control or EGR1 overexpressed Sclpfn1 BM cells were transplanted into CD45.1 recipients with competitors. Mice were treated with tamoxifen after 5 weeks. (G) Percentages of donor-derived CD45.2⁺ LT-HSCs were determined in BM and spleen (n = 3-4, *P < .05). (H) LT-HSCs were sorted from the BM of control or EGR1 rescued mice, and cell cycle was then measured by Hoechst 33342 and pyronin Y staining (n = 3-4, *P < .05). (I) Control and Sclpfn1 Lin⁻Sca-1⁺Kit⁺Flk2⁻CD34⁻ cells were analyzed for apoptosis by using Annexin V/7-AAD staining (n = 3-5; *P < .05). (J) LT-HSCs from the BM of control or EGR1 rescued mice were cultured for 8 days, and apoptosis was measured (n = 3-4; *P < .05).