Combined ectopic expression of PU.1 plus Id2 is insufficient to induce LCs. CD34⁺ cord-blood cells were combined transduced with vectors encoding PU.1-IRES-lyt2 and Id2-IRES-GFP, or with respective empty control vectors (CTRL). Cells were then stimulated in serum-free medium supplemented with the basic cytokine combination SCF/Flt3L/GM-CSF/TNFα with or without TGF-β1 as indicated. (A) Gated lyt2⁺GFP⁺ cells are analyzed for Langerin vs CD1a expression. Data are representative of 3 independent experiments. Numbers in quadrants indicate the percentage of cells in that quadrant. (B) Cells generated without TGF-β1 were sorted into GFP⁺ and GFP^- fractions (see representative FACS sorting region settings), or into lyt2⁺ and lyt2^- fractions. Sorted cells were analyzed for Id2, PU.1, and actin protein expression using Western blotting. (C) Double-transduced lyt2⁺/GFP⁺ cells were FACS-purified according to the sort region shown. Western blot analyses for Id2, PU.1, and actin are shown (n.s. indicates nonspecific).