Kt5720

l-Glutamine, fetal bovine serum, penicillin, streptomycin, amphotericin B, Hanks’ salts, trypsin, DMEM, and (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were from PanEco, Moscow, Russia. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), HEPES, DMSO, d-glucose, MPP⁺, KCN, MPP⁺, l-NAME, tert-butyl hydroperoxide, and bovine serum albumin were from Sigma-Aldrich, St. Louis, MO, USA. 666-11, SB 202190, SP 600125, salirasib, FIPI, KN-93, ML-193, PSB C5, HA-1004, U-0126, KT-5720, and U-73 were from Tocris Bioscience, Bristol, UK. Total RNA Purification kit was from Jena Biosciences, Jena, Germany. MMLV reverse transcription kit and qPCR master mix qPCRmix-HS SYBR were from Evrogen, Moscow, Russia. cAMP determination kit and BrdU cell proliferation assay kit were from Abcam, Cambridge, MA, USA. DNase I was from Thermo Fisher Scientific, Waltham, MA, USA.
The peptide was synthesized by methods of classical peptide chemistry in solution using both protected and free L-amino acids, as described earlier [49 (link)]. Purity of the final compound was not less 98% (HPLC analysis, Supplementary S2).

Wild type HEK293T-vector and HEK293T–EPAC1 cells were plated at 10,000 cells/well in 30 μl growth media on a corning Epic 384 well cell assay microplate (PerkinElmer), and then incubated overnight at 37 °C. Cells were then equilibrated for 2 h at room temperature and then a baseline measurement was recorded using an Enspire plate reader. The cells were then treated with varying concentrations of the EPAC1 antagonist, ESI-09 (Sigma Aldrich) or the PKA inhibitors, H-89 (Tocris) or KT5720 (Tocris), in the presence or absence of forskolin (Tocris) and rolipram (Tocris) or 007-(8-pCPT-O′-Me-cAMP) (Biolog). Dynamic mass redistribution measurements (DMR) were then taken every minute for 60 min. All treatments were made up in DMSO (Fisher Scientific) and diluted in 1 × HBSS (Life Technologies)/20 mM HEPES (Sigma Aldrich) assay buffer. For data analysis DMR readings from HEK293T-vector cells were subtracted from HEK293T–EPAC1 cells.

All drugs were obtained from Sigma-Aldrich unless otherwise indicated. IL-33 was obtained from Novus Biologicals. The ST2 neutralizing antibody was obtained from R&D System. The soluble form of ST2 (sST2) was obtained from Cohesion Biosciences. KT-5720 was obtained from Tocris. GS9973 and R406 were obtained from Selleck. JX-401 was obtained from Abcam. Stock solutions of KT-5720, AG490, R406, GS9973, LY294002, Akt inhibitor III, SB203580, SB202474 and JX401 were prepared in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the bath (maximum of 0.05%) had no functional effects on the currents measured.