Expression of a dominant negative form of HES1 (dnHES1) leads to up-regulation of Cdkn1 and Pten expression, and impaired T-cell development. (A) QRT-PCR analysis of mRNA expression of HES1 target genes (Cdkn1a,b,c, and Pten) in Rag2^−/− DN3 cells retrovirally transduced to express dnHES1 and/or GFP (MigR1), and then cultured with OP9-DL1 cells for 2 days before analysis. qRT-PCR results are normalized to β-actin expression levels, and the data are representative of 3 independent experiments. (B-C) Analysis of PTEN and phosphorylated GSK3β (Ser9) protein expression in (B) BWZ.36 cells or (C) DN3 cells retrovirally transduced to express dnHES1, shHES1, and/or GFP (MigR1) is shown as immunoblots of whole cell lysates probed with antibodies specific for PTEN, GSK3β (Ser9), or GAPDH. Data are representative of 3 independent experiments. (D-F) Developmental progression of FL-derived HPCs transduced to express dnHES1 and/or GFP (MigR1) and subsequently cultured for 10 days with OP9-DL4 cells. Flow cytometric analysis of (D) CD44 and CD25, and (E) CD4 and CD8, surface expression is shown for GFP⁺ gated cells on days 4, 7, and 10 of coculture as indicated, whereas panel F shows the corresponding cellularity of DP cells present in the cultures, as indicated. DP cellularity was obtained by multiplying the total cellularity by the percentage of DP cells present in the cultures of each independent experiment. Data are representative of 3 independent experiments.