4.7. Plasmids, siRNA Oligos and Transfection

For overexpression studies, the following plasmids (600 ng per transfection) were used: pFOXO1-EGFP-N1, pFOXO3-EGFP-N1, pFOXO6-EGFP-N1 and p-EGFP-N1 as empty vector control. pGL4.11-Bmal1::luciferase (200 ng) was used as circadian reporter, pFlag-CLOCK (600 ng) was used to rescue FoxO3 siRNA with pcDNA-Neo as empty vector control. For FoxO3 promoter studies, two plasmids with 1.4 kb and 2.1 kb of the FoxO3 promoter cloned into a pGL3::luciferase vector (see below) were used with pGL3::luciferase as empty vector control. For knockdown studies, siRNA oligos (50 pmol) against FoxO1 (siO1), FoxO3 (siO3), FoxO6 (siO6) or a scrambled siRNA (siC) were used. Transient transfections were performed using Lipofectamine2000 (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions unless specified otherwise.