Lipo8000

Human optic atrophy 1 (OPA1) promoter (−2000 bp) were cloned into pGL3-luciferase reporter vector (GenePharma Corporation). HTR8 cells were transfected with the pGL3-luciferase reporter vector and pRL-TK-Renilla-luciferase plasmid (Promega Corp) using Lipo8000™ for 12 h. Then cells were treated with cortisol (10⁻⁶ M) with or without CBX (10⁻⁶ M) for 24 h. Luciferase assays were measured by Dual-Luciferase^® Reporter Assay System (Promega).

293T cells were passaged 1 × 10⁵ per well in 48-well plate. In the following day, medium was changed and 0.6 µg of overexpression plasmids (pCMV-WRN/BLM, a gift from Dr. Vilhelm Bohr, NIA) were transfected using 0.8 µL Lipo8000 (Beyotime) in each well. After overnight incubation, medium was changed and transfected with 0.4 µg pGL3-basic luciferase plasmid (Promega) containing SHOX promoter sequences, 0.04 µg pRL-TK renilla plasmid and 0.6 µL Lipo8000 per well. Next day, cells were digested using Dual-Glo Luciferase Assay System (Promega) according to manufacturer’s protocol. Luciferase and renilla reading were taken by SpectraMax i3x (Molecular Devices) plate reader.