Gfi1b restricts proliferation and activation of HSCs. (A) Frequency of apoptosis of HSCs in the bone marrow (n = 3) of wt and Gfi1b-deficient mice was determined by flow cytometry (P ≤ .001 for both) using Annexin staining. (B) Mice were intraperitoneally injected with BrdU 18 hours before analysis. Bone marrow cells were stained for the indicated markers and for BrdU. A representative result from 3 independent examinations is shown. Mean values and SDs of the 3 independent experiments are depicted; P ≤ .05 for difference in cell cycle progression between wt and Gfi1b-deficient HSCs. (C) Bone marrow cells of pIpC-treated Gfi1b^fl/fl and MxCre tg Gfi1b^fl/fl mice were stained with the specific antibodies to define HSCs, Hoechst 3342 and verapamil according to manufacturer's instruction. Cells were then electronically gated to define HSCs (LSK, CD150⁺, CD48⁻), and Hoechst levels were determined. A histogram representative for 3 independent examinations is shown. (Bottom panel) Quantification of 3 independent experiments for HSCs and different MPP fractions; P ≤ .05 for difference in cell cycle progression between wt and Gfi1b-deficient HSCs. Values were obtained 30 days after the first (equivalent to 21 days after the last) pIpC injection. (D) Schematic outline to detect BrdU⁺ cells following published procedures. Forty percent of wt HSCs were qualified as “label retaining” whereas only 12% of Gfi1b^ko/ko HSCs still retained the label (BrdU; n = 4 for Gfi1b^fl/fl and n = 4 for MxCre tg Gfi1b^fl/fl; P ≤ .05). (E) Detection of reactive oxygen species (ROS) in HSCs. (Top panel) A representative result from 3 independent experiments is shown. (Bottom panel) Quantification of ROS levels in HSCs from animals with indicated genotypes (MFI, n = 3). Values were obtained 30 days after the first (equivalent to 21 days after the last) pIpC injection. (F) Frequency of HSCs in the bone marrow of wt (n = 7) and (n = 6) Gfi1b-deficient mice, which received N-Acetylcystein (NAC) or were left untreated (n = 14 for wt and Gfi1b deficient). Frequency of HSCs was determined by flow cytometry (P ≤ .01 between untreated and NAC-treated Gfi1b-deficient HSCs). Values were obtained 30 days after the first (equivalent to 21 days after the last) pIpC injection. (G) Frequency of HSCs in the spleen of wt (n = 3) and Gfi1b- (n = 4) deficient mice, which received NAC or were left untreated (n = 3 for wt and n = 5 Gfi1b-deficient) was determined by flow cytometry (P ≤ .01 between untreated and NAC-treated Gfi1b-deficient HSCs). (H) Frequency of HSCs in the peripheral blood of wt (n = 3) and Gfi1b- (n = 5) deficient mice, which received NAC or were left untreated (n = 6 for both genotypes), was determined by flow cytometry (P ≤ .01 between untreated and NAC-treated Gfi1b-deficient HSCs). (I) Genotyping of Gfi1b-deficient HSCs sorted from NAC and pIpC-treated Gfi1b-deficient mice. HSCs: genotyping of HSCs after treatment with NAC. NAC treatment did not affect excision of floxed Gfi1b exons and nonexcised HCSs were below detection level. CTL: Two controls with 1 sample consisting of cells with a flox/wt constellation and 1 sample consisting of wt cells.