Turner luminometer td20 20

The 3’-UTR of the SMAD4 gene was cloned into the 3’UTR of the OmicsLink^TM luciferase reporter vector (GeneCopoeia, Rockville, MD, USA). Mutagenesis was performed to generate reporter plasmids with mutations on miR-27a binding sites, as described in the reference [16 (link)]. HLECs were co-transfected with scrambled oligonucleotide, miR-27a mimic or inhibitor and OmicsLink^TM luciferase reporter vectors using Lipofectamine™ 2000. Twenty-four hours after transfection, luciferase activity was assayed with the Luc-Pair™ miR Luciferase Assay Kit (GeneCopoeia, Rockville, MD, USA) and a Promega Turner TD-20/20 Luminometer.
The plasmid P3TP-Lux was used to study the influence of miR-27a on the TGF-β signaling pathway and was kindly provided by Dr. Joan Massague (Memorial Sloan-Kettering Cancer Center, New York, NY, USA). HLECs were co-transfected with P3TP-Lux (1μg), pRL-TK (0.1μg), and different concentrations of miR-27a mimic, scrambled oligonucleotide or miR-27a inhibitor using Lipofectamine 2000. Twenty-four hours after the transfection, exogenous TGF-β1 (5 ng/ml) was added, and the luciferase assay was performed to measure the activity of firefly luciferase. Renilla luciferase activity was used for normalization.

Cellular ATP content was determined using a luciferase-based ATP assay kit (Molecular Probes, Cat. A22066). Cells were rinsed twice with cold PBS and 40 μL of cold 0.5% trichloroacetic acid (TCA, VWR. Cat. VW3928-2) was added and incubated on ice with shaking for 20 min. Cells were supplemented with 140 mL of 250 mM Tris-Acetate (pH 7.75) per sample, and 10 μL of this cell suspension was mixed with 90 μL of ATP Assay Solution (20 × reaction buffer, 0.1 M DTT, 10 mM Luciferin, 0.25 μL luciferase; Invitrogen). ATP levels were determined using a Turner TD20/20 Luminometer (Promega).