Microwin 2000

Forty-eight hours post-transfection, cells were washed once with phosphate-buffered saline (PBS) then incubated 1 h at 37°C in Tyrode buffer (137 mM NaCI, 0.9 mM KCI, 1 mM MgCI2, 1 1.9 mM NaHCO3, 3.6 mM NaH2PO4, 25 mM HEPES, 5.5 mM Glucose, and 1 mM CaCI2, pH 7.4). Cells were then treated with the ligands and prior to measurement treated with the substrate (5 min with 2.5 μM Coelantarazine 400a). Bioluminescence resonance energy transfer (BRET) was then measured between RlucII (BRET energy donor) and rGFP (enhanced bystander ebBRET acceptor)–tagged proteins. BRET values were read for 1 s per well using a Mithras™ LB940 Multimode Microplate Reader (Berthold Technologies, Bad Wildbad, Germany) for concentration–response curves. ebBRET values were obtained by calculating the ratio of the light emitted by the energy acceptor over the light emitted by the energy donor (donor 410 ± 70 nm/acceptor 515 ± 20 nm). Data were collected using the MicroWin 2000 software (Berthold Technologies, Bad Wildbad, Germany). They were then fitted and analyzed in GraphPad Prism (v9.0, GraphPad Software, Inc., San Diego, CA, USA).

HEK-293T cells permanently transfected with the EPAC sensor were split into 96-well plates at 15 to 20 × 10⁴ cells per well. After washing with PBS on the following day, PBS (containing calcium and magnesium salt) and coelenterazine solution were added to each well. After 10 min incubation, vehicle, curcumin (10 uM), resveratrol (10 uM) or both were added. The plate was then placed into a Mithras LB940 instrument (Berthold Technologies, Bad Wildbad, Germany) that allowed the sequential integration of the luminescent signals detected in the 465 to 505 nm and 505 to 555 nm windows using filters with the appropriate band pass and by using MicroWin 2000 software (Berthold Technologies). The BRET signal is determined by calculating the ratio of the light emitted at 505 to 555 nm to the light emitted at 465 to 505 nm.

Forty-eight hours post-transfection, cells were washed once with phosphate-buffered saline (PBS) then incubated 1 h at 37 °C in Tyrode buffer (137 mM NaCI, 0.9 mM KCI, 1 mM MgCI2, 1 1.9 mM NaHCO3, 3.6 mM NaH2PO4, 25 mM HEPES, 5.5 mM Glucose and 1 mM CaCI2, pH 7.4). Cells were then treated with the ligands and prior to measurement treated with the substrate (15 min with 2.5 µM Coelantarazine H for BRET¹ assays, and 5 min with 2.5 µM Coelantarazine 400a for BRET² and ebBRET assays). Bioluminescence resonance energy transfer (BRET) was then measured between RlucII (BRET energy donor) and either Venus (BRET¹ energy acceptor) or GFP₁₀ (BRET²) or rGFP (enhanced bystander ebBRET acceptor)–tagged proteins. BRET values were read for 1 s per well using a Tristar^®LB942 Multimode Microplate Reader (Berthold Technologies) and a Mithras™ LB940 Multimode Microplate Reader (Berthold Technologies) for kinetics and concentration–response curves, respectively. BRET¹ and ebBRET values were obtained by calculating the ratio of the light emitted by the energy acceptor over the light emitted by the energy donor (donor 480 ± 20 nm/acceptor 530 ± 20 nm for BRET¹ and 410 ± 70 nm/acceptor 515 ± 20 nm for ebBRET). Data were collected using the MicroWin 2000 software (Berthold Technologies). They were then fitted and analyzed in GraphPad Prism (v7.0, GraphPad Software, Inc).