Figure 4.
Mks direct TGFβ1 signaling in HSPCs but EP maturation is independently regulated. (A) Gating and representative flow cytometry data assessing Smad2/3 phosphorylation (pSmad2/3) within the LKS+SLAM population is shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice and for the FMO− control. Mean fluorescence intensity (MFI) of pSmad2/3 within the indicated hematopoietic populations is shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice (n = 7 per group). (B) Schematic showing TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice treated with 5 μg/kg TGFβ1 daily for 5 days and euthanized on day 6 for analysis. (C) MFI of pSmad2/3 staining is shown for Lin− HSPC subpopulations as quantified by flow cytometry of TGFβ1FL/FL (gray/light blue) and TGFβ1ΔMk/ΔMk (black/dark blue) mice treated with PBS or TGFβ1 (n = 3-4 mice per group). (D) RBC counts are shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice treated with PBS or TGFβ1 (n = 6-7 per group). The number of Ter119+annexin V+ apoptotic cells in marrow (E) and spleen (F) is shown for TGFβ1FL/FL or TGFβ1ΔMk/ΔMk mice (n = 4-9 per group). (G) Apoptosis within maturing EPs is shown for marrow cells after TGFβ1 treatment (n = 3-4 per group), as shown in Figure 3D. (H) Schematic showing C57BL/6J mice treated with either a TGFβ-neutralizing antibody (1D11) or isotype control antibody (13C4) at a dose of 10 mg/kg on days 1, 5, and 10 and then treated daily for 5 days with either PBS or EPO (300 U/kg). All mice were euthanized for analysis on day 16. (I) MFI of pSmad2/3 staining is shown for Lin− HSPC subpopulations as quantified by flow cytometry of mice treated with 13C4 control antibody (dark green/tan) or 1D11 (light green/red) and then PBS or TGFβ1 (n = 4 to 6 mice per group). (J) RBC counts are shown for mice treated with 13C4 or 1D11 followed by either PBS or EPO (n = 8 per group). (K) Representative data for apoptosis within maturing EPs is shown for marrow cells for B6 mice treated with 13C4 or 1D11 and then either PBS or EPO, as shown in Figure 3D. (L) The total number Ter119+ and the number of annexin V+ apoptotic Ter119+ EPs is shown for mice treated with 13C4 or 1D11 and then either PBS or Epo (n = 4-6 per group). All of the quantified data are shown as mean plus or minus SD (*P < .05, **P < .01, ***P < .001, or if not shown, the comparison was not significant). mAb, monoclonal antibody; MFI-RU, MFI relative fluorescence units.

Mks direct TGFβ1 signaling in HSPCs but EP maturation is independently regulated. (A) Gating and representative flow cytometry data assessing Smad2/3 phosphorylation (pSmad2/3) within the LKS+SLAM population is shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice and for the FMO− control. Mean fluorescence intensity (MFI) of pSmad2/3 within the indicated hematopoietic populations is shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice (n = 7 per group). (B) Schematic showing TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice treated with 5 μg/kg TGFβ1 daily for 5 days and euthanized on day 6 for analysis. (C) MFI of pSmad2/3 staining is shown for Lin− HSPC subpopulations as quantified by flow cytometry of TGFβ1FL/FL (gray/light blue) and TGFβ1ΔMk/ΔMk (black/dark blue) mice treated with PBS or TGFβ1 (n = 3-4 mice per group). (D) RBC counts are shown for TGFβ1FL/FL and TGFβ1ΔMk/ΔMk mice treated with PBS or TGFβ1 (n = 6-7 per group). The number of Ter119+annexin V+ apoptotic cells in marrow (E) and spleen (F) is shown for TGFβ1FL/FL or TGFβ1ΔMk/ΔMk mice (n = 4-9 per group). (G) Apoptosis within maturing EPs is shown for marrow cells after TGFβ1 treatment (n = 3-4 per group), as shown in Figure 3D. (H) Schematic showing C57BL/6J mice treated with either a TGFβ-neutralizing antibody (1D11) or isotype control antibody (13C4) at a dose of 10 mg/kg on days 1, 5, and 10 and then treated daily for 5 days with either PBS or EPO (300 U/kg). All mice were euthanized for analysis on day 16. (I) MFI of pSmad2/3 staining is shown for Lin− HSPC subpopulations as quantified by flow cytometry of mice treated with 13C4 control antibody (dark green/tan) or 1D11 (light green/red) and then PBS or TGFβ1 (n = 4 to 6 mice per group). (J) RBC counts are shown for mice treated with 13C4 or 1D11 followed by either PBS or EPO (n = 8 per group). (K) Representative data for apoptosis within maturing EPs is shown for marrow cells for B6 mice treated with 13C4 or 1D11 and then either PBS or EPO, as shown in Figure 3D. (L) The total number Ter119+ and the number of annexin V+ apoptotic Ter119+ EPs is shown for mice treated with 13C4 or 1D11 and then either PBS or Epo (n = 4-6 per group). All of the quantified data are shown as mean plus or minus SD (*P < .05, **P < .01, ***P < .001, or if not shown, the comparison was not significant). mAb, monoclonal antibody; MFI-RU, MFI relative fluorescence units.

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