Pcre luc

The human gene encoding OR10A3 was purchased from Novoprolabs (Cat# 746761-1; Shanghai, China) and subcloned into the pcDNA3.1 vector (Promega, Madison, WI, USA). Then, HEK293T cells were seeded into a 24-well plate (100,000 cells/well) and cultured for 24 h. The recombinant plasmid containing the OR10A3 was subsequently co-transfected into the HEK293 cells with (1) Renilla luciferase reporter plasmid (pRL-TK; Promega, Madison, WI, USA), (2) cAMP response element firefly luciferase construct (pCRE-luc; Promega), and (3) accessory proteins that facilitate the translocation of the olfactory receptor to the cell membrane (RTP1S, Ric8B, and Golf; gifts from Hiroaki Matsunami at the University of Duke in the USA and Cheil Moon at Daegu Gyeongbuk Institute of Science and Technology (DGIST) in Korea), using lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). 48 h after transfection, cells were treated with vehicle control or suberic acid (100 uM), incubated for further 6 h, and harvested for dual-luciferase assay using a Dual-Glo Luciferase assay system (Promega). The firefly luciferase signal was measured first and followed by Renilla luciferase in the same sample using a GloMax 20/20 luminometer (Promega).

HEK293T and Saos-2 cells were grown in DMEM (Thermo Fisher Scientific) supplemented with FBS (10% v/v). Twenty-four hours prior to transfection, cells were plated at 0.3 x 10⁵ cells/well in 96-well plates. Cells were transiently transfected with pRL-tK (2,5ng) and pCRE-Luc, NF-kB-Luc or pGL4.30 (NFAT-Luc, Promega) (25ng) along with 20ng of empty pcDNA3.1 vector, WT, L441R or W22X ZIP14 expression constructs using Fugene 6 (HEK293T cells) or ViaFect (Saos-2 cells) (Promega). Each transfection was carried out in triplicate and repeated independently in three separate experiments. Forty-eight hours after transfection, cells were lysed and firefly and renilla luciferase activity were measured on a Glomax Multi+ Luminometer (Turner Designs) using the dual luciferase reporter assay system (Promega). Finally, the ratio of the firefly and renilla luciferase measurement was calculated.