Dual light kit

B16-F10 cells were co-transfected with the NFκB-driven luciferase (Stratagene, La Jolla, CA, USA) vector and the Renilla reniformis luciferase reporter vector (Promega, Madison, WI, USA) at a ratio of 1:1/10 using Lipofectamine 3000 Reagent (Invitrogen; Carlsbad, CA, USA) for 4 h before being maintained with fresh medium for 24 h. Subsequently, cells were treated with MTII (10 nM) with or without lipopolysaccharide (LPS; 100 ng/mL) for 24 h. The NFκB-driven luciferase activities in cells were measured using a dual light kit (Promega, Madison, WI, USA) in Orion II microplate luminometer (Titertek Berthold; Pforzheim, Germany) and normalized with that of R. reniformis luciferase according to the manufacturer’s instructions.

Cells were co-transfected with HIF-1α-driven luciferase (Stratagene, La Jolla, CA, USA) vector and the Renilla reniformis luciferase reporter vector (Promega, Madison, WI, USA) at a ratio of 1:1/10 using Lipofectamine 3000 Reagent (Invitrogen; Carlsbad, CA) for 4 h before being maintained with fresh medium for 24 h. Subsequently, cells were treated with α-MSH (10 nM) for 24 h and then incubated in the presence or absence of CoCl₂ (100 μM) for another 24 h. The HIF-1α-driven luciferase activities in cells were measured using a Dual-Light Kit (Promega, Madison, WI, USA) in Orion II microplate luminometer (Titertek Berthold; Pforzheim, Germany) and normalized with that of R. reniformis luciferase according to the manufacturer’s instructions.