Expression from cyclin D3 promoter is regulated by Notch signaling. (A) CD4⁺ T cells after in vitro GSI pretreatment and from mice treated in vivo with GSI were stimulated with anti-CD3ϵ + anti-CD28 for 24 hours. Expression of cyclin D3 transcript was determined by RT-PCR after isolation of RNA. (B) Cyclin D3 luciferase reporter plasmid was transiently cotransfected with either 0.1 μg Notch1^IC expression plasmid or 0.1 μg of indicated Notch1^IC mutants⁵³  into 293T cells. Transfected cells were harvested 48 hours later for dual luciferase assay. The relative luciferase activity was calculated by normalizing against Renilla luciferase (pRL-CMV) activity that was used as an internal control. Values shown are representative of 3 separate experiments performed in triplicate. (C) ChIP of stimulated CD4⁺ T cells with or without GSI treatment. Anti-Notch1 (left panels) and anti-CSL (right panels) were used to precipitate protein complexes bound to DNA. Rabbit isotype control IgG was used as a negative control. Primers sets 1 (top panels) and 2 (bottom panels) were used in PCR to determine whether Notch1 and CSL are recruited to a specific region on cyclin D3 promoter.