Figure 4
Figure 4. Loss of Srf profoundly affects HSC engraftment ability. (A) Competitive and noncompetitive BMT assays. CD45.2 donor BM cells (4 × 106) isolated from Srf+/+;Mx1-Cre and SrfΔ/Δ;Mx1-Cre mice 5 weeks after pIpC treatment were transplanted into lethally irradiated CD45.1 congenic recipients. Donor cells were either equivalent in number to competitor CD45.1 BM cells (1:1) (left panel) or outnumbered competitor cells by 3-fold (3:1) (middle panel). Noncompetitive BMT were performed without competitor (1:0) (right panel). The percentage of donor chimerism (CD45.2) in the PB is given at the indicated time points posttransplantation (n = 4 mice per cohort). (B) Percentage of CD45.2 chimerism in BM subpopulations from the noncompetitive BMT 24 weeks posttransplantation. LSKs, MPs, megakaryocytes (Mks, CD41highCD42bhigh), myelo-monocytic cells (CD11b+Gr-1+). Values shown are mean ± SEM (n = 4 mice per cohort). (C) Short-term in vivo homing assay. LSKs at 5 × 104 and isolated from Srf+/+;Mx1-Cre and SrfΔ/Δ;Mx1-Cre mice (5 weeks after pIpC treatment) were stained with CFSE prior transplantation into lethally irradiated recipients. The percentage of CFSE+ LSKs present in the PB, BM, and spleen was scored by flow cytometry 24 hours after injection (n = 4 mice per cohort). The percentage of homing was determined as reported.28 (D) Absolute number of LSKs and MPs in total PB of Srf+/+;Mx1-Cre and SrfΔ/Δ;Mx1-Cre mice 35 days after pIpC treatment. Values shown are mean ± SEM (n = 5 mice per group, *P < .05). (E) Percentage of LSKs and MPs in spleen of Srf+/+;Mx1-Cre and SrfΔ/Δ;Mx1-Cre mice 35 days after pIpC treatment. Values shown are mean ± SEM (n = 5 mice per group, *P < .05 by Mann-Whitney test). (F) G-CSF mobilization assay on Srf+/+;Mx1-Cre and SrfΔ/Δ;Mx1-Cre mice 35 days after pIpC treatment. Mice were daily treated by G-CSF (200 μg/kg/d†) or NaCl during 5 days. At day 6, 100 μL PB were collected by retro-orbital plexus and cultured in methylcellulose medium. Scatter plots show the numbers of colonies scored after 7 days of culture (n = 4 mice per group, *P < .05 by Mann-Whitney test).

Loss of Srf profoundly affects HSC engraftment ability. (A) Competitive and noncompetitive BMT assays. CD45.2 donor BM cells (4 × 106) isolated from Srf+/+;Mx1-Cre and SrfΔ/Δ;Mx1-Cre mice 5 weeks after pIpC treatment were transplanted into lethally irradiated CD45.1 congenic recipients. Donor cells were either equivalent in number to competitor CD45.1 BM cells (1:1) (left panel) or outnumbered competitor cells by 3-fold (3:1) (middle panel). Noncompetitive BMT were performed without competitor (1:0) (right panel). The percentage of donor chimerism (CD45.2) in the PB is given at the indicated time points posttransplantation (n = 4 mice per cohort). (B) Percentage of CD45.2 chimerism in BM subpopulations from the noncompetitive BMT 24 weeks posttransplantation. LSKs, MPs, megakaryocytes (Mks, CD41highCD42bhigh), myelo-monocytic cells (CD11b+Gr-1+). Values shown are mean ± SEM (n = 4 mice per cohort). (C) Short-term in vivo homing assay. LSKs at 5 × 104 and isolated from Srf+/+;Mx1-Cre and SrfΔ/Δ;Mx1-Cre mice (5 weeks after pIpC treatment) were stained with CFSE prior transplantation into lethally irradiated recipients. The percentage of CFSE+ LSKs present in the PB, BM, and spleen was scored by flow cytometry 24 hours after injection (n = 4 mice per cohort). The percentage of homing was determined as reported.28  (D) Absolute number of LSKs and MPs in total PB of Srf+/+;Mx1-Cre and SrfΔ/Δ;Mx1-Cre mice 35 days after pIpC treatment. Values shown are mean ± SEM (n = 5 mice per group, *P < .05). (E) Percentage of LSKs and MPs in spleen of Srf+/+;Mx1-Cre and SrfΔ/Δ;Mx1-Cre mice 35 days after pIpC treatment. Values shown are mean ± SEM (n = 5 mice per group, *P < .05 by Mann-Whitney test). (F) G-CSF mobilization assay on Srf+/+;Mx1-Cre and SrfΔ/Δ;Mx1-Cre mice 35 days after pIpC treatment. Mice were daily treated by G-CSF (200 μg/kg/d†) or NaCl during 5 days. At day 6, 100 μL PB were collected by retro-orbital plexus and cultured in methylcellulose medium. Scatter plots show the numbers of colonies scored after 7 days of culture (n = 4 mice per group, *P < .05 by Mann-Whitney test).

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