Enspire multimode multiwell 96 plate reader

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).

Hep3B cells were seeded at a density of 8 × 10³ cells in 96-well dishes, serum-starved for 16 h, and then, when they had reached 80% confluency, were incubated with TGFβ1 (5 ng/ml) or T0901317 (5 μM), or a combination thereof for 48 h. Where indicated, Hep3B cells were seeded at a density of 1 × 10⁶ cells in 15 cm dishes and transiently transfected with the indicated siRNAs at a final concentration of 30 nM for 24 h, as described above. Afterwards, cells were seeded at the indicated concentration, serum-starved, and treated as reported. MTS assay was performed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Stockholm, Sweden) according to the manufacturer’s instructions. The MTS solution was diluted in a 1:10 ratio with cell media and the incubation was performed in dark for 2 h in a humidified incubator at 37 °C, 5% CO_2. The absorbance of formazan salts was read at 490 nm in an Enspire Multimode multiwell 96-plate reader (Perkin Elmer). Representative results of at least three biological replicates in technical triplicate are presented.