A740003

Synthesis and chemical details of C100814 are described in Additional file 1. Drugs were obtained from Sigma-Aldrich or, for MCC950 and A740003, from Tocris. Application of drugs in electrophysiogical experiments was via bath perfusion or locally via pressure ejection from a puffing pipette located at the slice surface (in the case of repeated brief ATP applications).

Key reagents and their sources were as follows: Escherichia coli LPS serotype 055:B5, TNF-α protease inhibitor-0 (TAPI-0) and ATP were from Sigma-Aldrich; selective p38 inhibitor (SB202190) was from Calbiochem Merck-Millipore; P2X7 receptor selective antagonists AZ10606120, A438079, and A740003 were from Tocris. The composition of the physiological buffer used in all experiments to stimulate macrophages with ATP was (in millimoles): 147 NaCl, 10 HEPES, 13 d-glucose, 2 KCl, 2 CaCl₂, and 1 MgCl₂; pH 7.4.
HEK293T cells (ATCC CRL-11268) were cultured in DMEM:F-12 media (1:1; Lonza) supplemented with 10% of fetal calf serum (Life Technologies) and 2 mM Glutamax (Life Technologies) and were routinely tested for mycoplasma contamination with a Mycoplasma Detection Kit (Roche). Lipofectamine 2000 (Life Technologies) was used according to the manufacturer’s instructions to transfect a plasmid coding for human TNF-α into HEK293T cells.

THP-1 cells were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 4.5 mg/ml D-glucose and 0.05 mM 2-mercaptoethanol at 37°C in 5% CO₂. Differentiation into macrophages was acquired by stimulation with 5 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h. GAS infection of THP-1 cells was performed as described above following 24 h priming with 5 ng/ml LPS. For inhibition of the P2X7 receptor, A-740003 (Tocris) was added 30 min before infection and was present throughout the experiment.

30 × 10³ Ma-Mel-19 or Sk-Mel-28 cells were cultured in RPMI-1640 medium and counted as described in ref. [14 (link)]. Whenever required, A740003 and AZ10606120 (Tocris Bioscience, UK) were added, respectively, at 20 or 2 μM.

Tissue culture media, antibiotics, fetal calf serum (FCS), and NP40 cell lysis buffer (10×) were purchased from Life Technologies (San Giuliano Milanese, Italy); lipopolysaccaride (LPS) (Ultra-Pure LPS-EB from E. coli 0111:B4 strain; only activates TLR4), Pam₃CSK₄, and ethyl-(6R)-6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate (CLI-095 or TAK 242) were from InvivoGen (Cayla-Invivogen Europe, Toulouse, France); A740003 from Tocris Bioscience, Pittsburgh, PA, USA); poly-L-lysine hydrobromide (mol wt 70,000–150,000), papain, DNase I (bovine pancreas), trypsin inhibitor, L-leucyl-L-leucine methyl ester (L-LME), protease inhibitor cocktail, Pefabloc® SC (100 mM), CU-CPT22, and all other biochemicals were purchased from Sigma-Aldrich (Milan, Italy) unless noted otherwise; recombinant human Apo-SAA (consensus SAA molecule corresponding to human Apo-SAA1α, except for the presence of an N-terminal methionine, the substitution of asparagine for aspartic acid at position 60, and arginine for histidine at position 71) from Peprotech (endotoxin level < 0.1 ng/μg protein; London, UK); QIAzol from Qiagen (Milan, Italy. Falcon tissue culture plastic-ware were purchased from BD Biosciences. Sterilin Petri plastic dishes (10 cm Ø) were obtained from Sarstedt (Verona, Italy).

Rat and mouse optic nerve head astrocytes were subcultured onto plastic 6-well plates and grown until confluent. Cells were incubated in control isotonic solution containing (in mM) 105 NaCl, 5 KCl, 4 NaHEPES, 6 HEPES acid, 1.3 CaCl₂, 5 glucose, 5 NaHCO₃ and 60 mannitol, pH 7.4 or in solution made 30% hypotonic solution by addition of dH₂0, for 4 h in the tissue culture incubator. Cells were pretreated with inhibitors Bay 11-7082 (4 μM), Brilliant blue G (BBG, 10 μM), A839977 (50 nM, Tocris Bioscience), A740003 (5 μM, Tocris Bioscience), carbenoxolone (10 μM, #C4790), probenecid (1 mM, #P8761) or ¹⁰Panx1 and scrambled peptide (100 μM, #3348 and #3708, respectively, Tocris Bioscience) for 1 h before adding test solutions. RNA was extracted immediately after the 4 h treatment.