Fig. 4.

GFP-GR nuclear movement is independent of lamin A/C under shear stress of 25 dyn/cm². A: live cell imaging of GFP-GR in BAECs transfected with lamin shRNA showed increasingly nuclear localized GR after 120 min of shear stress at 25 dyn/cm² (left). Similar GFP-GR movement was also observed in cells with control shRNA (right). Nuclei were labeled with Hoechst stain. Images were taken at ×40 magnification. Scale bar = 15 μm. B: quantitative image analysis of GFP-GR subcellular movement shows a 22.2 ± 1.8% and 24.1 ± 3.0% increase in nuclear brightness for lamin (n = 7) and control (n = 9) shRNA-treated BAECs respectively, after 120 min. C: Western blot of endogenous GR shows similar increase of GR in the nuclear (N) protein fractions compared with cytoplasmic (C) in both cell types following 120 min of shearing at 25 dyn/cm². Lamin A/C and TFIID served as the nuclear protein controls, and GAPDH was used as control for cytoplasmic protein. D: quantitative analysis of cytoplasmic and nuclear GR fractions based on repeated Western blots shows a significant increase of GR in the nuclear compared with cytoplasmic fraction following shear in both cell types (n = 3).