Luciferase reporter assay

The activation of NF-κB pathway was determined by using Cignal™ NF-κB luciferase Reporter Assay Kit (QIAGEN Ltd., Manchester, UK). HEK 293 or mBMSCs cells were cultured in 96-well plates and transfected with a mixture of NF-κB luciferase reporter negative control or positive control, along with Renilla construct (as an internal control) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 24 h, medium was changed with either CM-Adipo or CM-Control and cells were cultured for 24 h. Luciferase activities were determined using the Dual-Luciferase Assay System (Promega, Southampton, UK). Reporter activities were represented as arbitrary units after normalization to the internal Renilla reporter.