Expression of a dominant negative form of HES1 (dnHES1) or shRNA targeting HES1 (shHES1) in DN3a cells impairs T-cell differentiation across the β-selection checkpoint. (A) Developmental progression of DN3a cells transduced to express dnHES1 and/or GFP (MigR1) and subsequently cultured with OP9-DL4 cells for 2 to 4 days. Flow cytometric analysis of CD4 and CD8 surface expression is shown for GFP⁺ gated cells on days 2, 3, and 4 of coculture as indicated, with (B) the cellular fold expansion (total cellularity at each time point divided by the number of cells used at the start of the culture, input) observed in the cultures shown for the indicated times and conditions. (C) Developmental progression of DN3a cells transduced to express shHES1 and/or GFP (MigR1) and subsequently cultured with OP9-DL4 cells for 1 to 3 days. Flow cytometric analysis of CD4 and CD8 surface expression is shown for GFP⁺ gated cells on days 1, 2, and 3 of coculture as indicated with (D) the total cellularity observed in the cultures shown for the indicated times and conditions. Data are representative of 3 independent experiments, with standard deviation of the mean shown as error bars.