TGF-β signaling is highly active in dormant HSCs and regulates p57 expression. (A) Active TGF-β signaling in HSCs demonstrated by presence of phosphorylated Smad2/3 in the nucleus. Freshly isolated CD34⁻KSL HSCs (CD34⁻) and CD34⁺KSL hematopoietic progenitor cells (CD34⁺) were sorted in a serum-free culture-medium drop on slide glasses. The sorted cells were incubated at 37°C for 30 minutes in the presence or absence of TGF-β1 and then were stimulated with SCF and TPO for another 30 minutes. Freshly isolated CD34⁺KSL progenitor cells were also subjected to analysis. The cells were stained with DAPI (blue) and an anti–phospho-Smad2/3 antibody (red). Representative images are depicted on the top panels. In the bottom panels, the levels of immunofluorescence quantitated by an Olympus Laser Scanning Cytometer 2 (LSC2) are depicted. X- and y-axes are indicated in logarithmic and linear scales, respectively. The left panel presents data from freshly isolated CD34⁻KSL HSCs (CD34⁻) incubated for 30 minutes in the presence or absence of SCF and TPO. The middle panel presents data from freshly isolated CD34⁻KSL HSCs (CD34⁻) incubated for 30 minutes in the presence or absence of TGF-β1, and then stimulated with SCF and TPO for another 30 minutes. The right panel presents data from freshly isolated CD34⁻KSL HSCs (CD34⁻) and CD34⁺KSL hematopoietic progenitor cells (CD34⁺). (B) TGF-β up-regulates p57 gene expression in HSCs to induce cell-cycle arrest. Freshly isolated CD34⁻KSL HSCs were incubated in the presence of SCF (S) and TPO (T) for 12 hours, and were cultured for another 12 hours in the presence of TGF-β1 (TGF) in addition to SCF and TPO. The cells were stained with DAPI (blue) and an anti-p57 antibody (green). mRNA expression of mouse Cip/Kip genes was analyzed for each cell (bottom panel).