Constructs, transfection, and luciferase assay

The 963-bp 5′-flanking region of mouse Th was subcloned into pGL4.19-basic vector (Promega, Madison, WI, USA) using 5′-TTTGGTACCGTTTCCTTGGCTGAGGAAGCT-3′ and 5′-AACAAAGCTTAGTGCAAGCTGGTGGTCCCG-3′. A deletion construct of CRE was generated by deletion of the central four nucleotides (−45: TGACGTCA—> TGCA), as previously described^48,49 using the KOD mutagenesis kit (Toyobo). Constructs were confirmed by direct sequencing (ABI 3100; Applied Biosystems).

The HEK293T cells were plated in 96-well plates at a density of ~ 2 × 10⁶ cells per well in 125 µl DMEM. Th promoter-luciferase construct (133 ng) was co-transfected with Renilla luciferase (19 ng) using Xfect reagent (Takara Bio, Kusatsu, Japan). After 24 h, cells were washed with DMEM, and 75 µl of DMEM was added to each well. Modafinil (0.1 mM, dissolved in 0.1% DMSO/DMEM) was added into wells 12 h after washing⁵⁰. Sixteen hours later, luciferase activities were measured using the Dual-Glo luciferase assay (E2920; Promega) with SoftMax Pro (Molecular Devices, San Jose, CA, USA). Firefly relative luciferase activity was normalized against Renilla activity.