2.5. TOP-flash assay
3T3-L1 tsa hdac were plated into 96-well plates at a density of 2 × 104 cells/well. The cells were transiently transfected with 0.2 μg TOP-flash or FOP-flash (control). To normalize for transfection efficiency, a Renilla luciferase plasmid was used as an internal control. After 24 h, the cells were treated with adipogenic media in the presence or absence of TNF-α for 8 h. When indicated, an expression vector containing Msx2 or β-catenin was cotransfected. Luciferase activity was measured using a Dual-Glo luciferase assay kit. Relative luciferase activity was calculated after normalization to Renilla luciferase activity.
2.6. Msx2 knockdown using small interfering RNA (siRNA)
ON-TARGETplus SMARTpool siRNAs for mouse Msx2 and a non-targeting siRNA (control siRNA) were purchased from Dharmacon (Chicago, IL, USA). ON-TARGETplus SMARTpool siRNAs are a mixture of four siRNAs which increases both potency and specificity relative to a single siRNA. Transfection into 3T3-L1 cells was performed according to the manufacturer’s instructions.