American Society of Hematology

Figure 1

$Figure 1. Fbxw7α overexpression suppresses Fbxw7-target accumulation and HSC cell-cycle progression. (A-B) Quantitative PCR analysis of Fbxw7α (A) or Fbxw7β (B) transcripts in BM CD34− Flt3− LSK, CD34+ Flt3− LSK, CD34+ Flt3+ LSK, Lin−, or Lin+ fractions from 12-week-old mice. Each value was normalized to β-actin expression and is expressed as the fold induction compared with Lin+ samples (mean ± SD, n = 4). *P < .01. (C) Study design of Fbxw7α overexpression in LSK cells. LSK cells were transduced with pMY-IRES-EGFP (EGFP virus; control) or pMY-Fbxw7α-IRES-EGFP retrovirus (Fbxw7α virus). After 48 hours, EGFP+ cells were sorted and used for in vitro and in vivo assays. (D-F) EGFP+ cells were isolated from EGFP or Fbxw7α virus-transduced LSK cells. Isolated cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and anti-c-Myc, p53, or Notch1 antibody. Expression levels of c-Myc, p53, and Notch1 were quantified by the integrated fluorescence intensity (IFI) of the c-Myc (D), p53 (E), or Notch1 (F) signal normalized to the IFI of the DAPI signal in independent cells (mean ± SEM). (G) EGFP or Fbxw7α virus-transduced EGFP+ cells were stained with DAPI and antiphosphorylated S6 antibody. Phosphorylated S6+ cells were counted (mean ± SEM). (H) EGFP or Fbxw7α, β, or γ virus-transduced cells were harvested, and CD41−CD48− LSK cells were sorted and stained with DAPI and anti-Ki67 antibody. At least 450 cells/sample were counted (mean ± SD, n = 5). **P < .00007. (I-J) Short-term BrdU-labeling experiment with EGFP or Fbxw7α virus-transduced EGFP+ cells. Transduced cells were sorted and cultured on fibronectin-coated plates. After 42 hours of culture, cells were labeled for 3 hours with 10μM BrdU and stained with anti-BrdU antibody (green) and TOTO-3 (blue; I). Images were obtained and analyzed using a confocal laser-scanning microscope (FV1000; Olympus). UPIanApp 20×/0.70 objective lens (Olympus) and FV10-ASW2.0 viewer (Olympus). Scale bars represent 20 μm. Quantification of BrdU-labeled cells (J). At least 1500 cells/sample were counted (mean ± SD, n = 5).$

Fbxw7α overexpression suppresses Fbxw7-target accumulation and HSC cell-cycle progression. (A-B) Quantitative PCR analysis of Fbxw7α (A) or Fbxw7β (B) transcripts in BM CD34⁻ Flt3⁻ LSK, CD34⁺ Flt3⁻ LSK, CD34⁺ Flt3⁺ LSK, Lin⁻, or Lin⁺ fractions from 12-week-old mice. Each value was normalized to β-actin expression and is expressed as the fold induction compared with Lin⁺ samples (mean ± SD, n = 4). *P < .01. (C) Study design of Fbxw7α overexpression in LSK cells. LSK cells were transduced with pMY-IRES-EGFP (EGFP virus; control) or pMY-Fbxw7α-IRES-EGFP retrovirus (Fbxw7α virus). After 48 hours, EGFP⁺ cells were sorted and used for in vitro and in vivo assays. (D-F) EGFP⁺ cells were isolated from EGFP or Fbxw7α virus-transduced LSK cells. Isolated cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and anti-c-Myc, p53, or Notch1 antibody. Expression levels of c-Myc, p53, and Notch1 were quantified by the integrated fluorescence intensity (IFI) of the c-Myc (D), p53 (E), or Notch1 (F) signal normalized to the IFI of the DAPI signal in independent cells (mean ± SEM). (G) EGFP or Fbxw7α virus-transduced EGFP⁺ cells were stained with DAPI and antiphosphorylated S6 antibody. Phosphorylated S6⁺ cells were counted (mean ± SEM). (H) EGFP or Fbxw7α, β, or γ virus-transduced cells were harvested, and CD41⁻CD48⁻ LSK cells were sorted and stained with DAPI and anti-Ki67 antibody. At least 450 cells/sample were counted (mean ± SD, n = 5). **P < .00007. (I-J) Short-term BrdU-labeling experiment with EGFP or Fbxw7α virus-transduced EGFP⁺ cells. Transduced cells were sorted and cultured on fibronectin-coated plates. After 42 hours of culture, cells were labeled for 3 hours with 10μM BrdU and stained with anti-BrdU antibody (green) and TOTO-3 (blue; I). Images were obtained and analyzed using a confocal laser-scanning microscope (FV1000; Olympus). UPIanApp 20×/0.70 objective lens (Olympus) and FV10-ASW2.0 viewer (Olympus). Scale bars represent 20 μm. Quantification of BrdU-labeled cells (J). At least 1500 cells/sample were counted (mean ± SD, n = 5).

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