Luciferase reporter assay

Luciferase reporter assay was carried out following the procedure in Luciferase reporter assay user manual (Signosis, Inc.). The reporter vectors contain 4 repeats of the corresponding DNA binding sequences shown in gel shift assay section. In order to distinguish ATF4 and ATF6 activation, we cloned the reporter vectors for ATF4 and ATF6 with 5 repeats of specific consensus sequences for ATF4 and ATF6 respectively, TGACGTAAG [20] for ATF4 and TGACGTGG [21] for ATF6. The cells were first transfected with luciferase reporter vectors (Signosis Inc) for 16 h with Fugene 6 (Promega) in a 96-well plate, and then treated without or with 200nM TG and 10ug/ml TM for 6 h, 8 h and 16 h respectively. After removing the culture media and rinsing the cells twice with PBS, 200 μl of 1× cell lysis buffer was added to lyse the cells. After dislodging the cells by scraping them off from the plate, we transferred the cells to a 1.5-ml microcentrifuge tube before being centrifuged at 14,000 rpm at room temperature for 1 min to remove cellular debris. 10 μl of the cell extract was mixed with 50 μl of substrate (Signosis Inc), and luminescence was measured using a luminometer.