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Fig. 5. (A-D) MRTF translocation to the nucleus after force application. Representative images show NF (no force) and F (after 60 minutes of force). (Left panels, top rows) Endogenous MRTF A (red) in serum-reduced cells without force application was predominantly cytoplasmic. After force application endogenous MRTF A relocated to the nucleus. (Left panels, middle rows) Rat-2 cells transfected with MRTF-A–FLAG-tagged constructs for 20 hours, subjected to force and immunostained for FLAG. MRTF A (green) in serum-reduced cells without force application or with force application was predominantly nuclear. (Bottom left panels) Rat-2 cells transfected with MRTF-B–FLAG-tagged constructs for 20 hours. Nuclear translocation was never observed between 0 and 60 minutes. Cells were stained with DAPI for nuclear localization. (B) Percentage of cells in which transfected MRTF A was scored as predominantly cytoplasmic or predominantly nuclear after indicated times of force application. Results are given as the mean ± s.d. (C) Rat-2 cells transfected with MRTF A-FLAG (green). Blocking of ROCK with Y27632 (Y27632 F) and of actin filament assembly with latrunculin B (LB F) interfered with the nuclear translocation of MRTF A after force application. (D) Rat-2 cells transiently transfected with MRTF B-FLAG (green) were stimulated with serum for 30 minutes and show nuclear translocation of MRTF B.
