Sirius luminometer

GATA4-promoter was kindly provided by Professor Hyun Kook (Chonnam National University Medical School, Gwangju, South Korea). MSCs were plated in 24-well plates and transiently transfected with 1 μg of GATA4 promoter-luciferase reporter and 0.1 μg Renilla luciferase vector using X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland). Forty-eight hours later, cells were harvested, and luciferase activity was measured by using the Dual-Luciferase Reporter Assay (Promega, Wisconsin, USA) and a Sirius Luminometer (Berthold, Germany) according to the manufacturer's instructions.

Growth activity was examined by an ATP/luciferase-based assay (Vialight Plus kit, Lonza, Verviers, Belgium) as described [15 (link)]. Briefly, for measuring growth activity of adherent cells, 10⁴ siRNA-transfected cells were added to each well of a 24-well plate. For determining growth activity in 3D suspension cultures, the cells were incubated in ultra-low attachment 96-well microplates (Corning, Steinfurt, Germany) at a density of 5 × 10³ cells/well. Under both culturing conditions, cells were grown for four days before they were lysed and ATP-measured by the luciferase-based assay in a Sirius luminometer (Berthold). To study the effect of CAF-CM on growth, CAF-CM was added to the medium at a ratio of 1 to 5.

Cells were seeded at 80% confluency in a 60 mm culture dishes a day prior to drug treatment or transfection. Cells were harvested to measure intracellular O₂⁻. Cell pellets were lysed with 450 µL of ATP releasing reagent (Sigma Aldrich). Stock solution of 10 mM lucigenin (N, N’-dimethyl-9,9′-biacridinium dinitrate; Sigma Aldrich) was automatically injected into the samples immediately upon cell lysis using Berthold Sirius Luminometer (Titertek-Berthold). Luminescence emitted was then detected and measured every 0.6-second over a period of 14.4 seconds.

Transfection with a firefly luciferase reporter construct driven by the hypoxia response elements (HREs) of CA9, PGK and EPO was performed using the pLuc-MCS vector (Agilent Technologies, Inc., Santa Clara, CA, USA) and Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) as previously described (20 (link)). The HRE sequences are 5′-GGCTGTACGTGCATTGGAAACGAGAGCTG for CA9, 5′-TTTGTCACGTCCTGCACGACGCG for PGK and 5′-GGCCCTACGTGCTGTCTCACACAGCCTGT for EPO. A non-hypoxia-responsive plasmid, pRL-CMV (Promega Corporation), expressing Renilla luciferase was used as the internal control as described previously (20 (link)). Luciferase activities were determined using a Dual-Luciferase® Reporter Assay System (Promega Corporation) in a Sirius luminometer (Titertek-Berthold, Pforzheim, Germany), according to the manufacturer's instructions. Data are presented as the average ratio of firefly to Renilla luciferase activities [± standard deviations [SD)] from at least three independent experiments.

20 μl of cell extracts were employed for measuring luciferase activity by using Luciferase Assay System (Promega, Madison, WI) and a Berthold SIRIUS Luminometer (Oak Ridge, TN). First cells were co-transfected with a β-galactosidase expression vector, and then luciferase and β-galactosidase activity were measured. Initially luciferase activity was normalized to β-galactosidase activity and expressed as relative light units. Meanwhile, 2 μl of cell extracts were used for total protein concentration by Bio-Rad assay. Luciferase activity normalized to total protein from cell extracts produced similar results. In final results, the luciferase activity normalized to protein is reported as fold induction (fold change) compared to the time-matched control group in all experiments.