Fig. 3.

Disruption of actin filaments triggers nuclear transport of Neh2-GFP. (A) NIH 3T3 cells were stained with phalloidin conjugated with Texas red and 4′,6-diamidino-2-phenylindole (DAPI) after addition of DMSO (A; a vehicle control) or cytoskeletal filament disruptors, cytochalasin B (B), swinholide A (C), or colchicine (D). (E) subcellular localization of Neh2-GFP and Keap1 after treatment with cytochalasin B. Cells (4 × 10³) were transfected with expression plasmids of Keap1 (0.2 μg) and Neh2-GFP, a reporter protein of Nrf2 (0.8 μg). The latter is a fusion protein of Neh2 domain and GFP. Localization of these proteins was examined by fluorescence microscopy with use of GFP fluorescence and anti-Keap1 antibody, respectively (first and second rows). Merged images of Neh2-GFP and Keap1 signals are shown in the third row. Nuclei are shown with DAPI staining (fourth row). (Original magnification, ×400.) (F) nuclear transport of Neh2-GFP 3 h after the addition of cytochalasin B (Cyto-B), swinholide A (Swin-A), and colchicine (Col). Shown is the percentage of cells expressing Neh2-GFP in nucleus among the total transfected cells. The average and standard errors represent three independent transfection experiments.