Gata3 siRNA restores DN3-like cell development from E2A^−/− FL MPPs. (A) Flow cytometric analysis of E2A^+/+ or E2A^−/− FL MPPs transduced with control retrovirus or Gata3 siRNA retrovirus and cultured on OP9-DL1 in T cell–supportive conditions. GFP⁺ cells were enriched by cell sorting 48 hours after transduction and cultured for a further 12 days. (A) GFP versus side scatter (SSC) with gated regions indicate GFP^neg, GFP^low, and GFP^high populations. (B) CD44 and CD25 expression on GFP^neg, GFP^low, and GFP^high cells. One of 4 representative experiments is shown. (C) Average + SE of the percentage of cells expressing CD25 in all experiments. (D) qPCR analysis for Gata3, Id2, Ptcra, and Gfi1 mRNA in DN1-like and CD25⁺ cells isolated from E2A^+/+ or E2A^−/− cells transduced with control retrovirus (C) or with Gata3 siRNA (G). NA indicates not available. One of 2 similar experiments is shown. Bars represent the average + SE for the triplicate measurements in the experiment.