Signalling pathways of hypoxia- and 1,25-dihydroxyvitamin D
-stimulated leptin production. (A) Cells were treated with EtOH (−) or 1,25-dihydroxyvitamin D3 (VitD3; +) and immediately incubated under normoxia or hypoxia. Immunoblot illustrating hypoxia-inducible factor 2 (Hif-2) stabilization representative of three independent experiments is shown. (B) Cells were treated with scrambled silencing RNA (siScr) or siHif-1 for 24 hours, then with EtOH (−) or VitD3 (+) and immediately incubated under normoxia or hypoxia for 6 hours. Immunoblot illustrating Hif-1 stabilization representative of four independent experiments is shown. (C) Cells were treated with siScr or siHif-1 for 24 hours, then transfected with a Hif-1 response element driving Renilla luciferase gene and incubated under hypoxia for 36 hours. *P <0.05. (D) Cells were pretreated with vehicle (0.1% dimethyl sulphoxide) or different inhibitors for 1 hour under normoxia, then EtOH or VitD3 was added and cells were immediately incubated under hypoxia for 48 hours (n =5 experiments). PD98059, inhibitor of mitogen-activated protein kinase kinase (MEK); SB203580, p38 mitogen-activated kinase inhibitor; LY294002, Phosphoinositide 3-kinase inhibitor. *P <0.05. #Significant compared to vehicle (EtOH).