Regulation of endogenous PU.1 protein levels during TGF-β1-induced LC differentiation in serum-free medium. (A) Fluorescence-activated cell sorter (FACS) diagrams show representative CD34⁺ cells stimulated with “LC mix” (right, TGF-β1 plus SCF, Flt3L, GM-CSF, and TNFα) or LC mix without TGF-β1 (left) for 9 days analyzed for CD11b versus CD14 or Langerin versus CD1a. Bar diagrams represent the means and SEM of 4 independent experiments. The differences between -TGF-β1 and +TGF-β1 were significant (P = .022 for CD14⁺CD11b⁺, P = .020 for Langerin⁺CD1a^bright, paired 2-tailed t test). Microphotographs show typical LC cluster morphology by cells generated in LC mix (right). TGF-β1-nonsupplemented parallel cultures are compared (left). (B) The Western blot shows endogenous PU.1 or α-tubulin protein levels by CD34⁺ cells after 72-hour stimulation with the indicated cytokines. Lane 3 (center) and lane 5 (right) represent growth conditions giving rise to cells shown in the left and right FACS diagrams in panel A, respectively. Numbers in the quadrants of each diagram represent the percentage of cells in that quadrant. Data in panel A are representative of 5 independent experiments. Data in panel B are representative of 3 independent experiments. Cytokines: S indicates SCF (20 ng/mL); F, Flt3L (50 ng/mL); GM, GM-CSF (200 ng/mL); TNF, TNFα (2.5 ng/mL); and TGF, TGF-β1 (0.5 ng/mL).