Lfng overexpression augments binding of DL1 and DL4 by DN and DP thymocytes. (A) Binding of Fc, DL1-Fc, and DL4-Fc fusion proteins by HSC and DN thymocytes. Total fetal liver cells from E12.5-16.5 WT mice were stained with a cocktail of biotinylated lineage markers along with anti-CD117–APC and anti–Sca-1–FITC to identify Lin⁻ Sca-1^hi CD117^hi HSCs. Total thymocytes from WT mice were depleted of lineage-positive (Lin⁺) cells and stained with anti-CD117–APC and anti-CD25–FITC to identify DN1 (Lin⁻c-kit⁺CD25⁻), DN2 (Lin⁻c-kit⁺CD25⁺), DN3 (Lin⁻c-kit⁻CD25⁺), and DN4 (Lin⁻c-kit⁻CD25⁻) cells. Cells were subsequently stained with either human-Fc (control protein consisting of only the hinge and Fc portions of human IgG1; solid lines), DL1-Fc (dashed lines), or DL4-Fc (dotted lines) followed by anti–human IgG1-PE. (B) Effect of Lfng overexpression on binding of DL1-Fc and DL4-Fc in thymocyte subsets. Total thymocytes were stained with anti-CD4–FITC, anti-CD8–Alexa Fluor 633, and either human-Fc (control protein consisting of only the hinge and Fc portions of human IgG1), DL1-Fc, or DL4-Fc followed by anti–human IgG1-PE. Histograms compare binding of each protein to indicated thymocyte subsets from Lfng Tg⁺ (solid lines) versus WT (dashed lines) mice. Similar results were obtained in 3 independent experiments. (C) Lfng overexpression prolongs DL-induced Notch signaling in DN4 thymocytes. Sorted DN4 thymocytes from Lfng Tg⁺ and WT mice were cultured on OP9-DL1, and progeny were harvested and stained with anti-CD4–FITC, anti-CD8-PE, and biotinylated anti-CD25 (avidin Alexa Fluor 633) after 4 (top row) and 9 (bottom row) days of culture. Histograms display CD25 expression on DN and DP progeny from cultures initiated with Lfng Tg⁺ (solid line) versus WT (dotted line) DN4 thymocytes at each time point.