Neca 5 n ethylcarboxamido adenosine

Immortalized mouse mammary fibroblasts were generated in Dr. Harold Moses laboratory (Vanderbilt University), have been used in previous publications [46 (link)–48 (link)], and gifted to us. Primary mouse mammary fibroblasts were isolated from mouse mammary gland by FACS sorting as described below. Fibroblasts were grown in T-75 flasks (Fisher Scientific, USA) in DMEM medium (Gibco, USA) supplemented with 10% FBS, Antibiotic-Antimycotic (Sigma-Aldrich). For protein assessment, fibroblasts were grown in 10cm cell culture dishes (Corning, USA). When cells achieved 90–95% confluency, they were treated with TGFβ, 1ng/ml (R&D System, USA) and 0.01, 0.1, 1, 10, 100 uM NECA (5′-N-Ethylcarboxamido adenosine, Sigma-Aldrich, USA). Following inhibitors were used: for inhibition of A2a adenosine receptors, 1 uM SCH58261(Tocris, USA); for A2b receptors– 1 uM PSB603 (Tocris, USA); for PLC inhibition– 1 uM U73122 (Tocris, USA); for PKA inhibition—1 uM H89 (Tocris, USA); and for activation of adenylate cyclase– 10 uM forskolin (Sigma-Aldrich, USA). Human fibroblast cell lines IMR-90 (CCL-186), WS-1 (CRL-1502), WPMY-1 (CRL-2584), hTERT SMC PM151T (“hTERT” CRL-3291) and BJ (CRL-2522) were purchased from ATCC and cultured according to the manufacturer’s protocol.