TGF-β inhibits LRC and attenuates cytokine signals. (A) TGF-β inhibits cytokine-mediated LRC and maintains repression of the Akt-FoxO pathway in HSCs. Freshly isolated CD34⁻KSL HSCs were sorted into individual serum-free culture-medium drops 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. The cells were stained with DAPI (blue), CTxB (green), and an anti–phospho-Akt (red) or an anti-FoxO3a antibody (red). Proportions of CD34⁻KSL HSCs that showed lipid raft cluster formation are indicated as gray bars (Cluster+; right panel). (B) TGF-β inhibits activation of c-Src in HSCs. Freshly isolated CD34⁻KSL HSCs were incubated in the presence or absence of TGF-β1 for 30 minutes 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-c-Src antibody (red). (C) TGF-β maintains cytoplasmic accumulation of p57 in HSCs. Freshly isolated CD34⁻KSL HSCs were incubated in the presence or absence of TGF-β1 for 30 minutes 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-p57 antibody (green).