Xfect reagent

HeLa (human epithelial; ECACC no. 93021013) and HEK293T (human embryonic kidney SV40 transformed; ECACC no. 12022001) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum. 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/mL streptomycin were included in the culture media. All cells were maintained in a 5% CO₂ humidified atmosphere at 37 °C. For cell lysis, 2 × 10⁷ to 10⁸ cells per ml were incubated at 4 °C in NP40 buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 10% glycerol, 1% NP40, 1% aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/mL pepstatin, and 1 μg/mL leupeptin) for 20 min. The extract was centrifuged at 20000 g for 20 min, and the supernatant was stored at −80 °C. For transient transfection assays, 2–5 × 10⁶ HeLa cells/assay were resuspended in 200 μL of 15 mM HEPES-buffered serum-containing medium, mixed with 50 μL 210 mM NaCl containing 5–10 μg plasmid DNA and electroporated using a BTX Electrocell Manipulator 600 set at 240 V, 720 Ω, 950 μF. Cells were processed 24 h after electroporation. HEK293T cells were lipotransfected using the Xfect reagent (Takara Bio, Kusatsu, Japan) according to the manufacturer’s instructions and processed 48 h after transfection.

The FH variants (Figures 1B–D) were produced using the QuikChange XL site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). Each CFH cDNA clone was sequenced. The variants were transfected into human embryonic kidney 293T cells and DMEM was replaced with serum-free OptiMEM® (Invitrogen, NY). Transient transfections were performed using the Xfect reagent (Takara Bio USA, Mountain View, CA). Supernatants were collected after 72 h, concentrated 80×, and stored in aliquots at −80°C (9 (link)).

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.