Glomax multi multimode plate reader

Proximal promoters for the human Col1a1 (1.4 kb) and Col3a1 (1.5 kb) genes were generated from human genomic and subcloned into the pGL4.1[Luc2] vector. NIH-3T3 fibroblasts were seeded in 6-well dishes 24 h prior to transfection at a density that would yield ~ 60% confluency. Wells were co-transfected using jetPRIME DNA transfection reagent (Polyplus Transfection, Illkirch, France; #114–15) with 500 ng pGL4.1[Luc2] promoter–reporter plasmid and 500 ng expression vector for 48 h. Cells transfected with empty expression vector with the corresponding promoter construct served as background control samples. Promoter activity was assayed using a Luciferase Reporter Assay Kit I (PromoCell GmbH, Heidelberg, Germany; # PK-CA707-30,003–1) and luciferase activity was quantified on a GloMax Multi + Multimode Plate Reader (Promega, Madison, WI). All samples were assays in triplicates and relative promoter activity was normalized to total protein concentration for the corresponding sample.

NIH3T3 fibroblasts were co-transfected with scleraxis expression vector pcDNA-Scx or empty control vector pcDNA (pcDNA6, ThermoFisher Scientific, Canada), and pGL4.10-hGLS1 with or without E-box mutants using Lipofectamine 3000 (ThermoFisher Scientific, Canada) for 24 h. Renilla luciferase vector pRL was co-transfected as an internal transfection control. Luciferase activity was measured using the Dual Luciferase Reporter Assay System kit (Promega, USA) and a GloMax-Multi+ Multimode Plate Reader (Promega, USA) according to manufacturer’s directions.

Mouse cardiac fibroblasts were harvested after treatment with TGFβ₁, and the cell lysates were processed as per the manufacturer’s instructions (Glutamine/Glutamate-Glo™ Assay; J8021; Promega, USA) for determining the intracellular glutamine and glutamate concentrations. Two reactions were performed in duplicate per sample in an opaque, white 96-well plate (Corning Costar; 3912; Millipore-Sigma, Canada). For one reaction, the cell lysates were incubated with glutaminase enzyme to provide total glutamine + glutamate concentration; for the second reaction, the cell lysates were incubated without glutaminase enzyme, thereby providing the glutamate concentration alone. Subtracting these two values yielded the glutamine concentration. Luminescence was recorded using a GloMax-Multi+ Multimode Plate Reader (Promega, USA).