Fringe-mediated glycosylation increases Jagged2-induced Notch1 signaling. (A) Quantitative RT-PCR of LFNG expression during human T-cell development.^15,26  Data show the average of 3 independent sets of samples. Errors bars represent SEM. (B) Reporter assay of control and LFng-transduced U2OS Tet-on flp-in-Notch1 cells cotransfected with CBF-luciferase reporter plasmid pGL2-Gal4-luciferase and the normalizing plasmid pRL-TK expressing Renilla luciferase. After transfection, cells were cocultured with K562 expressing DLL1, DLL4, JAG1, JAG2, or control for 24 hours, and thereafter luciferase activity was measured. Bar graphs represent the average of 8 independent experiments. Errors bars represent SEM. (C) Left panels: Flow cytometric analysis of CD34⁺ Lin⁻ CB cells transduced with either control or LFng after coculture on OP9 cells expressing different Notch ligands (indicated above dot plot). Numbers in the dot plots indicate the frequency of CD34⁺CD7⁺ and CD7⁺CD5⁺ T-cell precursors. Right bar graphs: Average of the corresponding absolute cell numbers. Errors bars represent SEM. (D) Quantitative RT-PCR analysis of gene expression in human CD34⁺lin⁻ CB cells transduced with control or LFng, after 5 days of coculture on OP9-control (C), OP9-DLL1 (D1), OP9-DLL4 (D4), OP9-JAG1 (J1), or OP9-JAG2 (J2) stromal cells. Data show the ratio of expression in LFng versus control-transduced HPCs, and expression levels are normalized to β-actin. Data are the average from 2 sets of independent samples. Errors bars represent SEM.