S nitroso n acetylpenicillamine snap

Nitroglycerin was from American Regent (Shirley, NY). Rabbit polyclonal anti-eNOS/NOS 3, mouse anti-phosphorylated NOS3 (Ser 1177) and mouse anti-Caveolin-1 antibodies were from BD Biosciences (Franklin Lakes, NJ). Rabbit polyclonal anti-ubiquitin was from Abcam (Cambridge, MA). Protein A/G agarose bead was from Santa Cruz (Santa Cruz, CA). Dynabeads was from (Invitrogen Dynal, Oslo, Norway). Coumarin-7-boronate was synthesized in Dr. Balaraman Kalyanaraman's lab at Medical College of Wisconsin (Milwaukee, WI). Caveolin-1 scafolding domain peptide (CSD) and scrambled control peptides were from Calbiochem (San Diego, CA). L-NIO was from Cayman Chemical (Ann Arbor, MI). S-Nitroso-N-acetylpenicillamine (SNAP) and 4-hydroxyl-Tempol were from Sigma Aldrich (St. Louis, MO). Nucleofector kit was from Lonza (Walkersville, MD). Cav-1-YFP and empty YFP vector were provided by Dr. Rich Minshall at University of Illinois at Chicago. All other reagents are analytical grade or better.

The CFSE-labeled splenocytes were activated with ConA in the absence of MSCs and in the presence of 100 μM S-nitroso-N-acetylpenicillamine (SNAP) (Sigma-Aldrich, St.Louis, MO) in a 24-well culture plate. With the standard condition (MSC:SPC = 1:10), splenocytes were activated with ConA in the presence of 1 mM N(G)-nitro L-arginine methyl ester (L-NAME) (Sigma-Aldrich) or 200 μM 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) (Sigma-Aldrich). Splenocyte proliferation was evaluated by FACS.

Promastigotes at stationary phase were incubated at 5.10⁵ parasites/well in 96-well plates in the presence of different concentrations of S-Nitroso-N-acetylpenicillamine (SNAP) (Sigma-Aldrich), and N-acetyl-D-L-penicillamine (NAP) (Sigma-Aldrich) ranging from 25 to 100 μM. The plates were incubated at 26 °C for 24 h and the viability rate was determined by MTT assay. Briefly, 10 μl of MTT reagent (5 mg/ml) [3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyltetiazolium bromide] (Sigma-Aldrich) was added and incubated at 26 °C for 4 h. Solubilization of the formed formazan crystals was performed by using 100 μl of acidic isopropanol 0.04 N and 50 μl of DMSO. After incubation for 15 min at room temperature; the OD was measured at 570 nm using a BioRad Micro-plate Reader Photometer (BioRad Dynex, Washington, USA). The promastigote viability index was assessed as following: (absorbance of treated promastigotes/absorbance of control promastigotes) × 100. The 50% inhibitory concentration (IC₅₀), i.e. the SNAP concentration that decreases the growth by 50%, was calculated by regression analysis [21 ]. Results represent the average of 6 independent experiments.

Adult primary cultured cardiomyocytes were isolated from mouse hearts following the protocol described by Ackers-Johnson et al [40 (link)] and C166 mouse yolk sac endothelial cells were from American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells were incubated in the presence or absence of linagliptin (9 nmol/L) for 5 min for immunoblotting experiments and for 24 h for RNA sequencing. In addition, cardiomyocytes were incubated in the presence or absence of 100 µmol/L S-nitroso-N-acetylpenicillamine (SNAP) (Sigma-Aldrich, Oakville, Ontario, Canada) [41 (link)] for 5 min prior to immunoblotting. RNA isolation from cardiomyocytes was performed using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA). For co-culture experiments, adult primary cultured cardiomyocytes and C166 cells were cultured separately on opposite sides of Millicell 6-well plate inserts with a 10 µm pore size PET membrane (MCRP06H48, EMD Millipore, Billerica, MA, USA) at a 1:2 density under control conditions or with culture media supplemented with 9 nmol/L linagliptin for 5 min. Cardiomyocyte cGMP concentrations were determined by direct immunoassay (ab65356, Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions.

hCASMC were seeded in a 24 well plate in EGM-2 medium. After a 24-hour starving cells were wounded once with a small tip by scratching across the maximum diameter of each well. Pictures were taken immediately after scratching (time 0). Cell culture inserts containing EOCs were placed over the hCASMC. 30 minutes later, 0.1 μM PDGF (R&D Systems) or the nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP, Sigma Aldrich) was added as needed. pictures were also taken at 8 and 12 hours with EOC co-culture. Images were analyzed using Image J software by measuring the size of the denuded area.