Af 241 na

For urate priming experiments, adherent monocytes were primed for 24 h in RPMI supplemented with 10% human pool serum with or without urate (Sigma, 69-93-2) and recombinant TGF-β1 (R&D Systems, Catalogue number 7754-BH-005). After 24 h, cells were restimulated with 10 ng/mL ultra-pure E. coli LPS (InVivogen, Catalogue number tlrl-pelps). Subsequently, cell-free supernatants were collected. Secretion of cytokines was measured in supernatants using ELISA kits for IL-1β, IL-6, IL-1Ra and TGF-β (R&D Systems, Catalogue number DY201, DY206, DY280 and DY240 respectively).
To inhibit TGF-β receptor signalling, three inhibitors were used. The ALK4/5/7-kinase inhibitor SB-505124 (Sigma) in a concentration of 5 μM with DMSO as solvent control, 5Z-7-oxozeaenol (100 nM) dissolved in DMSO (Tocris Bioscience) and a blocking antibody against TGF-β receptor II (AF-241-NA, R&D systems) with mouse IgG1 as the isotype control (10 μg/mL). Cells were pre-incubated with the inhibitor for 0.5 h before adding urate.

Cells were lifted with Accutase® (Corning, Catalog # 25-058-CI) and stained with anti-human CD44-FITC (BD Pharmingen) and anti-human CD24-PB (Exbio) antibodies at manufacturer’s recommended dilutions in 0.1% BSA (Bovine serum albumin, Sigma) diluted in DPBS for 1 h in a rotary shaker at room temperature. Cells were analysed in the presence of propidium iodide (1 µg/mL) using a BD LSRFortessa (BD Biosciences). After doublet discrimination and compensation for spectral overlap, data were analysed by using FlowJo Software (BD Biosciences). For TGFβR2 surface expression, cells were stained with primary antibody (RandD Systems, Cat# AF-241-NA) as per manufacture recommended dilutions for 1 h and then with secondary goat antibody for 1 h.

The KPC tumor cells were cultured in KPC culture media containing 230 μmol/L RGD peptides (BML-P700-0005, Enzo Life Sciences) for 48 h before co-culturing. Mouse BMDMs were cultured in macrophage culture media containing 2.5 μg/μl anti-TGF-βRII antibodies (AF-241-NA, R&D System)^{39 (link)} for 24 h prior to polarization. Fresh antibody was added along with polarization cytokines for another 24 h during polarization. Then M1 macrophages were co-cultured with KPC PDA tumor cells or in monoculture for 24 h. In all, 230 μmol/L RGD peptides or 2.5 μg/μl anti-TGF-βRII antibodies were added to the culture media during co-culture, respectively. Paired M1 macrophages in monoculture were also cultured in media containing 230 μmol/L RGD peptides or 2.5 μg/μl anti-TGF-βRII antibody as control.