HEK293T cells were transfected with the following plasmids: FoxO1-WT (1,000 ng), FoxO1-ADA (1,000 ng), FoxO1-DN (1,000 ng), pCMV5 (1,000 ng), pGL3B (500 ng) or TH_long (500 ng), TH_medium (500 ng), TH_short (500 ng) and renilla (100 ng). After 24 h transfection, the activities of renilla and luciferase were determined using the Dual Luciferase Reporter Assay System (Promega, WI, USA). The luciferase activity was normalized to renilla activity. For FoxO1 knockdown experiments, cells were transfected with siRNA non-target control or FoxO1-specific siRNAs at final concentration of 100 nM together with pGL3B (500 ng) or TH_medium (500 ng) and renilla (100 ng) and luciferase assay was performed 48 h after transfection.
