Api 2

Recombinant mouse Shh N-terminus (1 μg/mL, R&D systems, Minneapolis, MN) or Purmorphomine (2 μM, Tocris Bioscience, 4551) were used to activate Hh signaling. FGF2 (10 ng/mL, R&D systems, Minneapolis, MN) was used to activate FGF signaling. BMP2 (100 ng/mL) was used to activate BMP2 signaling. PD173074 (1 μM, Tocris 3044) was used to inhibit FGFR. LY294002 (15 μM, Sigma, L9908) and API-2 (1 μM, Tocris 2151), were employed to inhibit PI3K and AKT respectively.

Recombinant mouse Shh N-terminus (1 μg/mL, R&D Systems, Minneapolis, MN, USA) or purmorphomine (2 μM, Tocris Bioscience, 4551) was used to activate Hh signaling. FGF2 (10 ng/mL, R&D Systems, Minneapolis, MN, USA) was used to activate FGF signaling. PD173074 (1 μM, Tocris, 3044) was used to inhibit FGFR. LY294002 (15 μM, Sigma, L9908), API-2 (1 μM, Tocris, 2151), rapamycin (0.5 μM, Selleckchem, S1039) were employed to inhibit PI3K, AKT, and mTOR, respectively.

OE33 OAC cells were cultured as described previously [8 (link), 32 (link)]. For proliferation experiments, cells were serum-starved for 24 h before stimulation with recombinant human leptin (1nM) (Bachem, UK), and then proliferation was assessed using a BrdU incorporation assay as previously described [20 (link)]. Apoptosis was induced by exposing serum-free leptin-treated cells with camptothecin (Merck, UK), and apoptosis quantified with the ApoPercentage assay as described previously [19 (link)]. We have previously confirmed that these assays provide comparable data to that obtained by cell counting and the MTT assay [8 (link), 9 (link)] and caspase-3 activity and quantification of intracellular nucleosomes [16 (link), 19 (link)]. For inhibitor studies, the Akt inhibitors, API-2 and GSK690693 (both 1 μm) [33 (link), 34 (link)] (Tocris, Abingdon, UK), were added 60 min prior to leptin. Cells were treated with adenoviral vector containing DN-Akt (Vector Biolabs, Pennsylvania, USA) 24 h prior to stimulation with leptin as described [26 (link)].