100 mm petri dishes

Caco2 cells were cultured in 100-mm Petri dishes (Sarstedt) in DMEM supplemented with 10% FBS and 1-mM sodium pyruvate (all purchased from Wisent), at 37°C in a 5% CO₂ atmosphere-humidified incubator. Transfections were carried out as cells were plated out in 6-well plates (Sarstedt). Approximately ∼1.2 × 10⁶ cells per well were plated and concomitantly transfected with 20-nM ASO and 4.5-μl Lipofectamine 3000 (Life technologies). After 24 h, cells were washed and scraped in ice-cold PBS. One fifth of the cells was used for RNA extraction (see the Quantitative PCR analyses section) and the rest for histone extraction with the EpiQuik™ Total Histone Extraction HTKit (Epigentek) according to manufacturer's protocol. Approximately 1 μg of total histones was fractionated through 12% acrylamide SDS-PAGE. Macroh2a1 polyclonal rabbit antibody (Active motif no39594) diluted 1000X was used for western blot analysis. Histone 3 monoclonal antibody (Active motif no61476) diluted 1000X was used as a loading control. Results are from three independent experiments, each performed in triplicate.

HEK293 cells were a kind gift of Dr. Laporte, McGill University^{57 (link)}. They were cultured in 100 mm Petri dishes (Sarstedt, Germany) at 37 °C and 5% CO₂ in the Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mm l-glutamine and 100 unit mL⁻¹ penicillin-streptomycin.
Transient transfections of vectors encoding BRET biosensors in combination with complementary signaling partners were performed in 100 mm Petri dishes (3 × 10⁶ cells) for G protein and Kir3.2 channel activation assays and in 96-wells culture plates coated with polyD-lysine (PerkinElmer, MA, USA) for βarr recruitment assays (32,000 cells/well), using the polyethylenimine transfection reagent (Polysciences, PA, USA) at a PEI/DNA ratio of 3:1^{58 (link)}. For cAMP production assays, stable cell lines expressing the GFP10-Epac-RlucBRET2-cAMP biosensor^{59 (link)} were plated in six-wells plates (Greiner bio-one, Austria) and stably transfected with 1 μg of either MORs or DORs (human or rat) biosensor using PEI. They were selected respectively using hygromycin (100 µg mL⁻¹) and puromycin (10 mg mL⁻¹).