M3763

Reagents were purchased and used as follows: GSK269962A (1 μM; Axon Medchem) in dimethyl sulfoxide (DMSO; #1167, Axon) was used as a ROCKi. LY-294002 (10 μM; #1130, Tocris Bioscience) in DMSO was used as a PI3K inhibitor. Adenosine (5 μM; A4036, Sigma-Aldrich) in DMSO, APCP (10 to 100 μM; M3763, Sigma-Aldrich), and anti-CD73 antibody (clone 2C5, Medimmune/Astra-Zeneca) were used. Time of treatment and drug concentration were specified in each experiment.

The CD73 inhibitor adenosine 5′-(α,β-methylene) diphosphate (200 uM, M3763 Sigma-Aldrich), adenosine (10 uM, A9251 Sigma-Aldrich), TGF-β (5 ng/ml, T7039 Sigma Aldrich) and TGF-β inhibitor (1 uM, S4317 Sigma Aldrich) were added freshly to the culture media daily over 3 weeks.

RAW-Blue cells (a murine RAW 264.7 macrophage reporter cell line) were purchased from InvivoGen. This reporter cell line stably expresses a secreted embryonic alkaline phosphatase gene inducible by NF-κB activation that can be detected. The assay was performed according to the manufacturer’s instructions. Briefly, 2 × 10⁴ RAW-Blue cells were incubated with 20 µg of GBex alone or in combination with (1) NF-κB pathway inhibitor (IKK16, 200 nmol, 2539, Tocris), (2) anti-TGF-β Ab (IDII, 100 nM, obtained from Dr Andrew Hinck, University of Pittsburgh), (3) COX inhibitor (Celecoxib, 1 µM, PZ0008-, Sigma), (4) anti-IL-2R Ab (10 µg, MAB224, Millipore), (5) CD73 inhibitor (Adenosine 5′-(α,β-methylene)diphosphate, 1 µM, M3763, Sigma), (6) phosphodiesterase inhibitor (Rolipram, 1 µM, R6520, Sigma), (7) anti-IL-6 Ab (10 µg, mabg-hil6-3, Invivogen), (8) anti-FAS Ab (10 µg, 05-338, Millipore), and (9) anti-TNF-α Ab (10 µg, htnfa-mab1, Invivogen) for 24 h under standard conditions. After incubation, 20 µL of media was collected and incubated with 200 µL QUANTI-blue reagent (Invivogen) and optical density was measured at 655 nm using TECAN spectrophotometer (TECAN).