Foxp3 Suppresses NF-κB-Dependent Transcription

<div><p>(A and B) HEK 293T cells (5 × 10⁵) were seeded into six-well plates 1 d prior to transfection with an NF-κB luciferase reporter vector (200 ng) in the presence or absence of indicated concentrations of a Foxp3 expression vector or a control vector and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity (A) and Foxp3 mRNA expression (B).</p><p>(C) HEK 293T cells (5 × 10⁵) were seeded into six-well plates 1 d prior to transfection with an NF-κB luciferase reporter vector (200 ng) in the presence or absence of a Foxp3 expression vector or a control vector (1,500 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24, 48, 72, and 96 h posttransfection and analyzed for luciferase activity.</p><p>(D) CD4⁺ T cells (2 × 10⁶) from three healthy donors were nucleofected with an NF-κB luciferase reporter vector (1,000 ng) and Foxp3 or control expression vectors (2,000 ng) and internal reference plasmid (1,000 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity shown in A, C, and D was normalized to the internal reference control.</p>