Mrs2768

All general chemicals were purchased from Sigma-Aldrich, except those indicated specifically. Phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, pluronic acid F-127 were from Life Technology, and MRS2768, 5-BDBD, NF546 and NF340 from Tocris Bioscience. MRS2768 was also obtained from Santa Cruz. Huh-7 cells were kindly provided by Prof M Harris (University of Leeds, UK).
Huh-7 and HepG2 cells were maintained in DMEM supplemented with 10% heat inactivated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C and 5% CO₂, and passaged when cells reached 70% confluency. Huh-7 and HepG2 cells were transfected with 15 nM small interfering RNA (siRNA) targeting the P2YR11 gene with the following sequences (siP2Y11): forward 5’-CCUGCUGGGCAGCGUCAUC(TT)-3’ and reverse 5’-GAUGACGCUGCCCAGCAGG(TT)-3’. Irrelevant sequences, not targeting any known gene were used as control sequences (siCTL): forward, 5’-GCCGACCAAUUCACGGCCG(TT)-3’ and reverse, 5’-CGGCCGUGAAUUGGUCGGC(TT)-3’. These sequences were produced by Sigma-Aldrich. Transfection was performed with Lipofectamine RNAi max (Invitrogen) according to the manufacturer's instructions, and cells were used 72 hr after transfection.

Fibroblast migration was analyzed as previously described [74 (link)]. A wound was created through the cell monolayer using a 200 µL pipette tip. When required, cultured cells were then incubated with Panx1 channel blockers: 200 µM probenecid (PBN) (Thermo Fisher Scientific, Waltham, MA, USA), 100 µM ¹⁰Panx mimetic peptide, or scrambled mimetic peptide ¹⁰Panx (Sc ¹⁰Panx) 100 µM (Tocris, St. Louis, MO, USA). The wound areas were measured for 24 h, and standardized digital images were acquired every 2 h using a phase-contrast Nikon Eclipse TE200-U microscope (Nikon Instruments, Melville, NY, USA). Image analysis was performed using ImageJ (version 1.49v; NIH, MD, USA). Wound area  =  100% − percentage of the initial wound area size.
The purinergic receptor antagonists used here were: 10 µM A-740003 (Tocris, MO, USA), 100 µM suramin (Sigma-Aldrich, St. Louis, MO, USA), 200 µM Brilliant Blue G (BBG, Sigma-Aldrich, Louis, MO, USA), 10 µM AR-C118925XX (Tocris, St. Louis, MO, USA), 5 µM MRS-2768 (Tocris, Louis, MO, USA), and 1 mM ATP (Sigma-Aldrich, Louis, MO, USA). Purinergic receptor antagonists and agonists were incubated for 30 min before inducing the wound and maintained for the experiment’s duration.