Firefly luciferase assay kit

HepG2 cells were seeded at a density of 18 × 10³ cells in 24-well dishes. Cells were transfected with a human SNAIL promoter construct encompassing approximately 900 bp fused to the luciferase cDNA [33 (link)] or the proximal promoter of the LXRα target gene ATP binding cassette transporter A1 (ABCA1) fused to luciferase, together with pCMV-β-galactosidase plasmid, the latter used as reference control, at final amount of 100 ng per plasmid DNA. Transient overexpression was performed with the indicated plasmids, while transient silencing was conducted with the indicated siRNAs at a final concentration of 30 nM. Transfection was performed when cells were ~80% confluent for 48 h, as explained above. Where indicated, cells were subsequently serum-starved for 16 h and then TGFβ1 (5 ng/ml) or T0901317 (5 μM), or a combination thereof were added for the indicated time periods. Luciferase assay was performed using the Firefly Luciferase Assay Kit (Biotium, Techtum, Stockholm, Sweden) according to the manufacturer’s instructions. β-Galactosidase activity was assessed via quantification of conversion of its substrate o-nitrophenol-glucose at 420 nm, and used as control. Chemiluminescent and colorimetric readings were performed in an Enspire Multimode multiwell 96-plate reader (Perkin Elmer, Upplands Väsby, Sweden).

Fly lysate was prepared from from 2- to 3-day-old adult males and assayed using the Firefly Luciferase Assay Kit (Biotium) following the manufacturer's instructions. See Supplementary Materials and Methods for details.

PC3 cells were transiently transfected with the GRN-Luc reporter plasmid containing a 1.8-kb genomic fragment encompassing the human GRN promoter cloned upstream of firefly luciferase. Cells were cotransfected with the Renilla luciferase plasmid for normalization and to determine transfection efficiency. After 48 h, cells were treated individually or in combination with 50 nM progranulin or 100 µM LCA for 6 h. Luciferase activity was assayed using firefly luciferase assay kit and Renilla luciferase assay kit from Biotium. The data from three independent experiments were normalized on Renilla luciferase.

TZM-bI cells were seeded at 10,000 cells per well in 100 μL medium in 96-well black plates with half area transparent bottom (Greiner Bio-One). Each well was inoculated with 100 μL of viruses that were subjected to 10-fold serial dilution and infection was done in triplicate. After 48 h, viral infection was determined by a Firefly Luciferase Assay Kit (Biotium). Briefly, after removal of media, 20 μL of 1x Firefly Lysis Buffer were added to each well. After 5 m, 15 μL of Firefly Luciferase Assay Buffer 2.0 containing 0.3 μL of D-luciferin at 10 mg/ml were added to each well and luciferase activities were measured by Veritas Microplate luminometer (Turner Biosystem). Viral infectivity was calculated by normalizing luciferase activities with the amounts of p24^Gag in viral inoculum.