Knockdown of Pten expression in Rag2^−/− DN3 cells with TCRβ allows for T-cell differentiation across the β-selection checkpoint in the absence of Notch signals. (A) qRT-PCR analysis (normalized to β-actin) of Pten mRNA expression in Rag2^−/− DN3 cells transduced to express PTEN shRNA (LMP-HP_522 or LMP-HP_524) or firefly luciferase shRNA (LMPff). Data are representative of 3 independent experiments. (B) Analysis of PTEN protein expression in NIH3T3 cells retrovirally transduced to express the indicated shRNAs is shown as immunoblots of whole cell lysates probed with antibodies specific for PTEN or GAPDH. Data are representative of 3 independent experiments. (C-D) Developmental progression of culture-derived Rag2^−/− DN3a cells transduced to express shRNAs as indicated and subsequently cultured for 6 days with OP9-DL1 or OP9-Ctrl cells. (C) Flow cytometric analysis of CD4 and CD8 surface expression is shown for GFP⁺ (shRNAs) and YFP⁺ (TCRβ or MIY) gated cells, whereas panel D 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.