Fig. 1SIRTUIN1 interacts with CLOCK and BMAL1 in cell nuclei. (A) Schematic diagram of Venus-based bimolecular fluorescence complementation (BiFC) constructs. (B) Expression of SIRT1-N-terminal of Venus (VN) was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis separation followed by immunoblotting with an anti-green fluorescent protein (GFP) antibody (arrows). (C) Results from BiFC analysis and immunocytochemical experiments (ICC). COS7 cells were coexpressed with SIRT1-VN and either CLOCK-C-terminal of Venus (VC) or BMAL1-VC. Cells were immunostained with anti-GFP, anti-CLOCK, and anti-BMAL1 antibodies (red) to detect SIRT1-VN, CLOCK-VC, and BMAL1-VC, respectively. Nuclei were visualized with 4',6-diamidino-2-phenylindole (DAPI; blue). The images were acquired by fluorescence microscopy using specific filter sets for yellow fluorescent protein and red fluorescent protein. Scale bar=10 µm.
Fig. 2Identification of the SIRTUIN1 (SIRT1)-binding domain of BMAL1. (A) Schematic diagram of mouse BMAL1. (B) Binding of SIRT1 to BMAL1 deletion mutants was analyzed by bimolecular fluorescence complementation (BiFC). COS7 cells were transfected with plasmids encoding SIRT1-N-terminal of Venus (VN) and BMAL1-C-terminal of Venus (VC) wildtype (WT) or its various mutants (Δnuclear localization sequence [NLS], BMAL1 without a functional nuclear localization signal; Δbasic-helix-loop-helix [bHLH], encoding BMAL1 Δ71 to 140; ΔPer-Arnt-Sim [PAS]-A, BMAL1 Δ210 to 320; ΔPAS-B, BMAL1 Δ350 to 480; Δtranscriptional activation domain [TAD], BMAL1 Δ553 to 625). The images were captured by fluorescence imaging microscopy using specific filter sets for yellow fluorescent protein and red fluorescent protein. Scale bar=10 µm. ICC, immunocytochemical; DAPI, 4',6-diamidino-2-phenylindole.
Fig. 3Interaction of SIRTUIN1 (SIRT1) with CLOCK and BMAL1 upon resveratrol treatment. (A) COS7 cells were transfected with expression plasmids encoding SIRT1-N-terminal of Venus (VN) and either CLOCK-C-terminal of Venus (VC) or BMAL1-VC. Twelve hours after transfection, cells were treated with 100 µM resveratrol for 6 hours and then imaged. Scale bar=20 µm. (B) Bimolecular fluorescence complementation (BiFC) signals from control and resveratrol-stimulated groups were compared with signals from 4',6-diamidino-2-phenylindole (DAPI)-stained cells. Values are expressed as mean±SEM % (n=10 fields for each group). Veh, vehicle; RSV, resveratrol; YFP, yellow fluorescent protein.
Fig. 4The effects of resveratrol on Ebox-Luc and Per1-Luc transcriptional activities. NIH3T3 cells expressing wild-type CLOCK, BMAL1, and either (A) Ebox-Luc or (B) Per1-Luc promoters were treated with increasing amounts of resveratrol (10 and 100 µM) to determine transcriptional activity. Luciferase activities in cell extracts were analyzed and normalized by cotransfected pRL-TK activity in each sample. To examine the effects of SIRTUIN1 (SIRT1) and resveratrol on the transcriptional activity of (C) Ebox-Luc and (D) Per1-Luc, SIRT1 was expressed with or without CLOCK and BMAL1 in NIH3T3 cells and incubated in resveratrol (10 and 100 µM). Values are expressed as mean±SEM in arbitrary units (AU), where the mean activity of the empty vector was set at 1 (n=3). aP<0.01 vs. vehicle-treated group.