Zardaverine

HEK-293T cells were transfected with cDNAs encoding AT₁R, AT₂R, or MasR in the presence or the absence of ACE2-HA. Cells were serum-starved in DMEM alone for 2 h before initiating an experiment. Starved cells were detached and suspended in culture medium containing 50 µM of the phosphodiesterase inhibitor, zardaverine (Sigma-Aldrich). Cells were then distributed into 384-well microplates at 2500 cells/well and stimulated for 15 min with increasing concentrations of agonists, including Ang II for AT₁R, CGP for AT₂R, and Ang 1-7 for MasR. Agonists were added at concentrations 0.1 nM to 3 µM or vehicle alone; this was followed by the addition of 0.5 µM forskolin or vehicle alone for an additional 15 min. Readings were performed after a 1 h incubation at 25 °C. Homogeneous time-resolved fluorescence energy transfer (HTRF) measurements were performed using the Lance Ultra cAMP kit (PerkinElmer, Waltham, MA, USA). Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab Technologies, Offenburg, Germany).

CL316243, isoproterenol, resveratrol, rosiglitazone, amlexanox, zardaverine, H89, IBMX, and thiazolyl blue tetrazolium blue (MTT) were from Sigma. [1-¹⁴C] palmitic acid was from Moravek Biochemicals. 2′3′-cGAMP and 2′3′-cGAMP control were from Invivogen. ABT-737, Q-VD-OPh and nigericin were from Cayman Chemical. Digitonin was from EMD Millipore. Antibodies to UCP1 (Cat# Ab23841), C/EBPβ (Cat# Ab32358), ChREBP (Cat #157153), Prdm16 (Cat# ab106410), and ERP57 (Cat# Ab10287) were obtained from Abcam; antibodies to cGAS (Cat# 31659), p-TBK1 (Cat# 5483), TBK1 (Cat# 3013), p-IRF3 (Cat# 4947), IRF3 (Cat# 4302), STING (Cat# 13647), PKA Substrates phosphorylation (Cat# 9621), p-HSL (Cat# 4139), HSL (Cat# 4107), PPARγ (Cat# 2443), ATGL (Cat# 2439), FASN (Cat# 3180), p-ACC (Cat# 3661), ACC (Cat# 3662), and SCD1 (Cat# 2794) were all from Cell Signaling; an antibody to PGC1α (Cat# ST1204) was from EMD Millipore; an antibody to TNFα (Cat# CG1601) was from Cell Applications, Inc; an antibody to Complex IV (Cat# A6403) was from Molecular Probes; Lamin A antibody (Cat# 3267-100) was from BioVision; antibodies to beta-tubulin and myc were made from monoclonal antibody-producing cell lines obtained from ATCC; A polyclonal anti-DsbA-L antibody is homemade from rabbit and also available in EMD Millipore (Cat# ABS1644).