RUNX3 overexpression rescues LC differentiation in PU.1cKO. (A) BM cells of the indicated genotypes were transduced with either empty vector (RV-Empty) or a RUNX3 encoding retrovirus (RV-Runx3). Dot plot represents the expression of DEC205 and EPCAM in transduced (GFP⁺) or nontransduced (GFP⁻) CD11c⁺MHCII⁺ DCs 48 h after infection. Numbers indicate the percentage of cells in each quadrant. Graph shows the mean proportion ± SD of LCs generated with empty vector or Runx3 retrovirus. Data are representative of six independent experiments. *, P < 0.05; **, P < 0.01 (unpaired Student’s t test) for the indicated comparisons. (B) Alignment (produced in clustalW) of the Runt domain from RUNX1 and RUNX3. Predicted DNA binding motifs are boxed (from Prosite). Arrowheads indicate the critical residues for RUNX1 DNA binding (as in Li et al., 2003). Gray shadowed residues indicate the position of the single mutation directed in RUNX3 construct. (C) The R196Q mutant of RUNX3 was generated by site directed mutation, and its functionality to promote LC differentiation was assessed by flow cytometry as in A. This experiment is representative of three independent experiments. Numbers indicate the percentage of cells in each quadrant.