Inhibition of Notch signaling does not affect Runx1, Runx3, or Cbfb expression. (A) Flow cytometric analysis of Lin⁻ FL cells cultured on OP9-DL1 in the absence and presence of the indicated concentrations of GSI for 7 days. CD8⁻CD19⁻Gr-1⁻GFP⁻ cells were analyzed for CD44 and CD25 expression (n_experiments = 8). (B) Gene expression profile of DN1 cells sorted from day 7 OP9-DL1 cocultures treated with dimethyl sulfoxide (□), 1.0 μM GSI (), or 3.0 μM GSI (■). Cell sorting, RNA preparation, and real-time PCR were performed as described for Figure 4A. Taqman probes were used for the quantification of Runx1, Runx3, Cbfb, and Hprt, and SYBR green was used for the remainder of the genes. Expression of each gene was quantified in comparison to a standard curve prepared with dilutions of spleen cDNA. The expression of individual genes is displayed relative to Hprt. Data are averaged from 4 independent experiments. Error bars represent SEM. *indicates significant difference (P≤ .05) between GSI-treated and untreated cells. (C) Runx1 expression in lymphoid progenitors (c-kit⁺CD27⁺CD25⁻) isolated from day 3 OP9-DL1 cultures in the absence and presence of 3 μM GSI (averaged from triplicate samples from 3 independent experiments). The increase in Runx1 expression in GSI-treated cultures was significant at P < .01. (D) Ectopic expression of Runx1 in Cbfb^+/+ FL cells cultured on OP9-DL1 in the absence and presence of GSI at 3 μM. Analysis was performed as in panel A.