White opaque 96 well microplate

Luciferase activities were measured using the Dual-Glo Luciferase Assay System (Promega). At 48 h post-transfection, medium was removed and cells were lysed using 50 μL of luciferase reagent diluted with 50 μL of PBS. After a 10-min shaking incubation, 90 μL of cell lysate from each well was transferred to a White Opaque 96-well microplate (PerkinElmer). Firefly luminescence was measured at 25 °C on a DTX880 Multimode Detector (Beckman Coulter) (MDA-MB-468 and MDA-MB-231 cells), or CLARIOstar (BMG LABTECH) (BT549 cells). Renilla luminescence was measured for each well 10 min after adding 45 μL of Stop&Glo reagents.
The relative luciferase activity of each construct was calculated as the ratio of firefly: Renilla luminescence, averaged amongst the triplicates and normalised to the positive control.
For each gene, luciferase assays were replicated in three independent experiments. Relative luciferase activities were normalised to the mean wild-type activity across the replicates for each gene. Using R, the relative luciferase activities of TE-deleted constructs were compared against the wild-type with a one-tailed t-test assuming equal variances, to test our hypothesis that the deletions would reduce promoter activities. P < 0.05 indicated statistical significance.

The sorted APCs were seeded in 96-well plates at 5 × 10⁴ cells per well in 200 μl of growth media. Following overnight incubation, the cells were exposed to a concentration of 10 ng/ml of TGF-β1, along with fresh growth medium, for a duration of 72 h. To initiate the assays, the growth medium was carefully removed from each well and replaced with 100 μl of PBS. Subsequently, 50 μl of mammalian cell lysis solution was added to lyse the cells, and the resulting cell lysate was transferred to a White Opaque 96-well Microplate (Perkin Elmer, Villebon-sur-Yvette, France) in preparation for the ATP assay. The intracellular ATP content was quantified using the ATP-lite assay kit (Perkin Elmer, Villebon-sur-Yvette, France). Luminescence intensity emanating from each well was measured using a FLUOstar Optima plate reader (BMG Labtech, Offenberg, Germany).

Coelenterazine was purchased from Carbosynth. Linear polyethylenimine (PEI, 25 kDa) was supplied by Polyscience. FuGENE® HD Transfection Reagent, furimazine (NanoGlo®), HaloTag® NanoBRET™ 618 ligand and T4 DNA ligase were from Promega. Phosphosite-specific antibodies pS355/pS356-β2 (Cat. no.: 7TM0029A) and pT360/pS364-β2 (Cat. no.: 7TM0029B) were from 7TM Antibodies, and the antibody against pS261 (Cat. no.: PA5-12977) was from Invitrogen. Anti-HA-tag antibodies were purchased from Cell Signaling Technology. Hanks balanced salt solution (HBSS), Dulbecco’s modified Eagles’s medium (DMEM), Lipofectamin 2000, HA-beads, TetraSpeck fluorescent beads, 96-well white polystyrene LumiNunc microplates, all antibiotics and pertussis toxin (PTX) were from Thermo Fisher. The HTRF cAMP accumulation kit and the DERET substrate, SNAP-Lumi4-Tb, were purchased from Cisbio. SNAP-Surface Alexa Flour 488, SNAP-Surface Alexa Flour 549, SNAP-Surface Alexa Flour 649, Q5® High-Fidelity polymerase, NotI-HF®, ApaI, anti-SNAP-tag antibody (Cat. no.: P9310S) were obtained from New England Biolabs (NEB). Effectene was from Qiagen. All other chemicals and compounds were from Sigma Aldrich. TopSeal-A PLUS, Dihydroalprenolol Hydrochloride ([³H]-DHA, 250µCi, 9.25MBq), WGA PVT 500 MG SPA Beads, and OptiPlate-96, and White Opaque 96-well Microplates were from Perkin Elmer.

1.5 × 10⁴ GH3 cells per well were seeded into white opaque 96-well microplates (PerkinElmer), serum starved for 24 hours in media containing 1mM luciferin, then stimulated in triplicate as indicated. Microplates were covered with Breathe-Easy sealing membranes (Sigma) and Luc activity of wells measured using a FLUOstar Omega (BMG Labtech) over a 24-hour period with cells maintained at 37°C with 5% CO₂-95% air. Photon counts for each well were integrated over 5 seconds and repeated every 15 minutes. Results are shown as mean fold induction relative to an unstimulated control ± SD of at least 3 independent experiments. For statistical analysis, P values were calculated using Student's t test.