Pgl4.19 basic vector

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 (link),49 (link)} 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 × 10^{6 (link)} 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 washing^{50 (link)}. 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.

HEK293T cells were grown in 48-well cell culture plates for 24 h. They were then transiently transfected with a promoterless luciferase vector (pGL4.19-basic) (Promega, United States) or with a pGL4.19-basic vector with the LBX1 promoter fragment (−2,060 to −120) harboring the rs1322330 A-allele or G-allele (construct rs1322330_A-allele or construct rs1322330_G-allele). The cells were transfected with 660 ng of pGL4.19 (with or without insert) along with 33 ng Renilla plasmid. Lipofectamine 2000 (Invitrogen, United States) was used for transfection into HEK293 cells, according to the manufacturer’s protocol. Cells were harvested at 48 h after transfection, and luciferase assays were then performed with the Dual-Luciferase Assay Kit (Promega, United States) according to the manufacturer’s instructions. Cell lysates were tested first for firefly luciferase activity and then for Renilla luciferase activity. Firefly luciferase luminescence values were divided by Renilla luciferase luminescence values from the same transfection to control for differences in transfection efficiency.