H 89 dihydrochloride hydrate

Rabbit polyclonal MEF2A antibody was produced with the assistance of the York University (Toronto, ON, Canada) Animal Care Facility. MEF2D (BD Biosciences, Mississauga, ON, Canada, 610775), KLF6 (Santa Cruz, Dallas, TX, USA, R-173 and E-10), Actin (Santa Cruz, sc-1616), IRDye 680RD goat anti-rabbit (LI-COR, Bioscience, Lincoln, NE, USA) and IRDye 680RD goat anti-mouse (LI-COR, Bioscience) were used for Immunoblotting experiments. FITC- and TRITC-conjugated α-rabbit and α-mouse secondary antibodies and 4,6-diamidino-2-phenylindole (DAPI; D9542), H₂O₂ (H0904) and H89-dihydrochloride hydrate (B1427) were purchased from Sigma Aldrich (Toronto, ON, Canada). Atenolol (Sigma Aldrich, A7655), Isoproterenol hydrochloride (Sigma Aldrich, 1351005) and ICI 118551 hydrochloride (Abcam, Toronto, ON, Canada, ab1200808) were purchased for use in cell culture.

Wistar rats were bred in our Laboratory Animal Breeding Centre, Department of Toxicology, Poznan University of Medical Sciences (Poznan, Poland). The animals were kept under standardized light conditions (14:10 h light/dark cycle, illumination onset at 6.00 a.m.) at 23 °C, 50–60% air humidity, 8–10 air changes per hour (mechanical, via HEPA filters) and maintained on a standard diet with free access to tap water. Animals were decapitated between (11–12 a.m.) and studied organs were promptly removed. The number of rats, their sex, age, and body mass used in the current study are given in the descriptions of the individual experiments. The study protocols were approved by the Local Ethics Committee for Animal Studies in Poznan (protocols No. 11/2015 and 75/2016, 06/March/2015). ACTH (Synacthen) was purchased from Novartis (Basel, Switzerland), testosterone (Testoviron-Depot) from Schering AG (Berlin, Germany), estradiol (Estradiol-Depot) from Jenapharm (Jena, Germany) and H-89 dihydrochloride hydrate (Catalog Number B1427) and hCG (Chorionic gonadotropin human Catalog Number CG10, lyophilized powder) from Sigma-Aldrich (St. Louis, MO, USA). Other reagents, unless stated otherwise, were purchased from Sigma-Aldrich (St. Louis, MO, USA) or from Avantor Performance Materials Poland S.A. (Gliwice, Poland).

Cells were treated with 50 μM forskolin (Sigma-Aldrich) or 30 μM H89 dihydrochloride hydrate in DMSO (Sigma-Aldrich) for 30 min before QD-labeling and imaging. 10 μM calcium ionophore A23187 in DMSO (Sigma-Aldrich) was added to pre-labeled cells for 5 min before imaging and 5 mM methyl-β-cyclodextrin (MBCD) in water was added to cells for 15 min before labeling and imaging.

The preparation of methanolic H. stoechas extract was previously described [12 (link)], and a plant voucher was deposited at the Universidad San Jorge herbarium (ref. 002–2014). The phenolic profile of this extract was previously determined [12 (link)] (see Introduction).
The compositions of the buffers used were as follows: Krebs buffer (in millimolar): NaCl 120, KCl 4.7, CaCl₂ 2.4, MgSO₄ 1.2, NaHCO₃ 24.5, KH₂PO₄ 1, and glucose 5.6. Ca²⁺-free Krebs: NaCl 120, KCl 4.7, CaCl₂ 2.40, MgSO₄ 1.2, NaHCO₃ 24.5, KH₂PO₄ 1, glucose 5.6, and ethyleneglycoltetraacetic acid (EGTA) 1. Ca²⁺-free high K⁺ Krebs ([K⁺]_o = 50 mM). All buffers were adjusted to pH 7.4. Apamin (AP), arzanol, barium chloride dihydrate (BaCl₂), glibenclamide (Glib), heparin (H), H-89 dihydrochloride hydrate, indomethacine, Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), phenylephrine (PE), ruthenium red (RR) and verapamil (V) were obtained from Sigma (Madrid, Spain).
Tetraethylammonium (TEA), 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) and Rp-8-Br-PET-cGMPS were purchased from Tocris (Madrid, Spain). AP was diluted in acetic acid. Glib, TRAM-34 and arzanol were dissolved in dimethyl sulfoxide (DMSO). All other chemicals were dissolved in distilled water.