Figure 3. Regulation of DC differentiation by Wnt pathway.

A. Enriched HPCs were cultured with GM-CSF with or without 500ng/ml Wnt5a recombinant protein for 5 days and the phenotype of cells was evaluated by flow cytometry. Left panel – typical example of one experiment. Right panel –Cumulative results of three performed experiments are shown. B. Allogeneic MLR. Cells from experiments in panel A were co-cultured with T cells isolated from allogeneic BALB/c mice for 4 days at different ratios. Cell proliferation was measured in triplicate by [³H]-thymidine uptake. Values are the mean ± SE. C. NIH-3T3 cells were transfected with control vector or β-catenin shRNA. Whole cell lysates were prepared 72 h after transfection and proteins were analyzed in Western blot. D. Enriched HPCs were transfected with GFP, β-catenin-GFP, or β-catenin-shRNA-GFP vectors. Cells were cultured in CCM with 20 ng/ml GM-CSF for 5 days and evaluated by flow cytometry. CD45⁺GFP⁺ cells were gated for further analysis. Left: Typical example of one experiment. Right: Cumulative results of three performed experiments. E–H. Lin⁻ c-kit⁺ myeloid progenitor cells were sorted from bone marrow of β-catenin^flox+ and wild-type β-catenin^flox− mice. Cells were then infected with either control retrovirus or retrovirus containing a Cre construct and cultured with a cocktail of cytokines to generate mature myeloid cells. E. The level of β-catenin in transduced cells was evaluated 3 days after infection using qRT-PCR or Western blotting (inset). F. The proportion of DCs was evaluated by flow cytometry in myeloid progenitors 5 days after infection with Cre-retrovirus. G. Allogeneic MLR. Cells 5 days after Cre-retrovirus were co-cultured with T cells isolated from allogeneic BALB/c mice for 4 days in different ratios. Cell proliferation was measured in triplicate by [³H]-thymidine uptake. Values are the mean ± SE. H. Myeloid progenitor cells (2×10⁴ β-catenin^flox+, CD45.2⁺ cells) infected with control or Cre retroviruses were transferred into lethally irradiated congenic (CD45.1⁺) mice together with 10⁶ syngenic (CD45.1⁺) bone marrow cells. Cells were evaluated in spleens 3 weeks later. CD45.2⁺CD45.1⁻ donors cells were gated and the phenotype was evaluated by flow cytometry.