Arl67156

ATP levels were detected using bioluminescence by combining samples with recombinant firefly luciferase and its substrate d-luciferin (ATP Determination Kit, Life Technologies, Cat. No. A22066). The ATPase inhibitor (ARL67156, 30 μM, Sigma-Aldrich) was added to the medium to minimize breakdown of ATP. Samples were exposed to CNO (10 μM) for 5 minutes, then the supernatant was collected, and samples read immediately using a FilterMax F5 plate reader at 28°C. ATP measurements were expressed relatively to control samples.

Before moxibustion intervention, 50 μl of ATPase (125 μg/ml, 250 μg/ml, 500 μg/ml, Sigma) or ARL67156 (62.5 μg/ml, 125 μg/ml, 250 μg/ml, Sigma) was injected (intramuscular injection) into ST36 acupoint. ATPase, known as adenosine triphosphatase, is an enzyme that catalyzes the hydrolysis of ATP to ADP and free phosphate ions. It can promote ATP degradation. ARL67156 (N⁶-diethyl-β, γ-dibromomethylene-ATP trisodium salt hydrate) is a nonspecific ecto-ATPase inhibitor. It was firstly described as the ecto-ATPase inhibitor in 1995 [33 (link)] and demonstrated to inhibit ecto-ATPase activity in various tissues of rats [34 –37 (link)].

The enzymatic activity of CD39 and CD73 was evaluated based on the percentage of hydrolysis of ATP or AMP respectively by HPLC. Briefly, Th17 cells generated in vitro were diluted in Hanks’ balanced salt solution (HBSS) and incubated in a 96- well flat-bottom plates at 0.5x10⁵ cells/well with 10 μM ATP (Sigma-Aldrich) in the presence or absence of the CD39 inhibitor ARL67156 (Sigma-Aldrich) at a concentration of 50 μM or with 10 μM AMP (Sigma-Aldrich), with or without the CD73 inhibitor APCP (Adenosine 5’-(a,b-methylene) diphosphate) (50 μM) (Sigma-Aldrich). After 1 h, the cells were harvested, transferred to ice for 15 min, and then centrifuged at 1000 x g for 10 min. Supernatants were collected and stored at -20°C until further analysis. HPLC analysis was carried out in a Water Breeze system using an anion exchanger column (Mono Q; GE Healthcare, Chalfont St Giles, UK). The mobile phase used consisted of a linear gradient from buffer A (Tris–HCl 100 mM, pH 7.8) to buffer B (Tris–HCl 100 mM, NaCl 1 M, pH 7.8). The effluent was monitored at 257 nm using an online UV detector. The column was calibrated using ATP, AMP and adenosine as standards.

Nano-SiO₂ and nano-TiO₂ were purchased from IoLiTec (Heilbronn, Germany), and nano-ZnO is a gift from Dr. Amir Yazdi (Lausanne, Switzerland). Nanoparticles were sonicated for 30 min and used at a concentration of 125–500 μg/ml in vitro or at a concentration of 5 mg/kg in vivo, as mentioned. A740003 is a gift from Dr. F. Rassendren (Montpellier, France). adenosine deaminase (ADA) (A5168), ADP, ADPβS, adenosine (Ado), apyrase grade VII (A6535), ARL67156, ATPγS, ATP, carbenoxolone (Cbx), DPCPX, flufanemic acid (FFA), inosine (Ino), MRS1523, MRS1754, MRS2395, MRS2578, periodate-oxidised ATP (oATP), phorbol 12-myristate 13-acetate (PMA), SCH58261 and U73122 were from Sigma (St. Quentin Fallavier, France); suramin was from VWR (Fontenay-sous-bois, France); LPS (lipopolysaccharide from Escherichia coli, serotype 055:B5) was from Invivogen (Toulouse, France); 2-APB, 5-Iodotubercidin, Forskolin, MRS2500, NBMPR and Z-YVAD-fmk were from Tocris (Bristol, UK) and NECA and SQ22536 were from Merck Millipore (Nottingham, UK).

All cell culture reagents were obtained from Cultilab (Brazil). Suramin, ARL 67156, and gadolinium chloride were purchased from Sigma Aldrich (St. Louis, MO).

PGE₁ was from Biomol (Hamburg, Germany), anti-phospho-VASP (Ser157) antibody was from Cell Signaling (Danvers, MA, USA), Adenosine diphosphate (ADP) was from Enzo Life Science (Lörrach, Germany), 2-thiouridine 5′-triphosphate (2S-UTP), 4-thiouridine 5′-triphosphate (4S-UTP), 4-thio-isobutyl uridine 5′-triphosphate (4S-ib-UTP) was from Jena Bioscience (Jena, Germany), Collagen was from Nycomed (Linz, Austria), uridine triphosphate (UTP), UTPγS, and ARL 67156 were from Sigma (Steinheim, Germany). All other chemicals were of the best available quality, usually analytical grade.