In vitro generated LCs require PU.1 but not ID2. (A) BM cells from the indicated genotype were cultured in the presence of GM-CSF and TGF-β. At day 3, CD11c⁺MHCII⁺ cells were monitored by flow cytometry for their expression of DEC205 and EPCAM. A representative plot for each genotype is shown. Numbers represent the frequency of CD11c⁺MHCII⁺ cells generated for the indicated genotype. Bar graphs show the mean proportion ± SD of in vitro generated LC from at least four experiments per genotype. ***, P < 0.001 (unpaired Student’s t test) compared with the wild-type sample. Data from IRF4-deficient (IRF4 KO) cells are from Irf4^−/− mice. (B) BM cells from the indicated genotype were cultured in the presence of GM-CSF and TGF-β. At day 3, CD11c⁺MHCII⁺ cells were sorted and DNA extracted. The deletion efficiency was assessed by PCR. Cre⁻, cells derived from the respective fl/fl Itgax^cre+/+ mice; H₂O, no DNA added to the reaction. The position of the wild-type (wt), loxP flanked (fl), and deleted (Δ) alleles are shown for each genotype. Data are representative of three independent experiments.