Physiology software

Nuclear translocation of Nrf2 was assessed by western blotting in vivo and by both western blotting/immunofluorescent staining in vitro. By using a cell lysis buffer system (Santa Cruz Biotechnology), the whole‐cell extract from cultured RAECs and harvested thoracic aorta tissue was prepared. Cytoplasmic protein and nuclear protein were extracted with Cytoplasmic Extraction Reagents (Pierce Biotechnology Inc, Rockford, IL) and Nuclear Extraction Reagents (Pierce), respectively. Protein concentrations were determined by a BCA protein assay kit (Pierce). The protocol of western blotting is described in the below paragraph. Cultured RAECs were fixed and incubated with primary antibody against Nrf2 (1:250; Cell Signaling Technology) at 4°C for 8 hours. Then, the secondary antibody conjugated with Alexa 488 (Invitrogen) and DAPI was used to incubate the cells. Quenching of fluorescence was alleviated with SlowFade Lignt Antifade kit (Molecular Probes). Samples were exited at 519 nm and observed at 442 and 495 nm with an inverse fluorescence microscope (Axio Imager 2; Carl Zeiss, Jena, Germany). Fluorescent intensity was determined by using Zeiss Physiology software (Ver.3.2; Carl Zeiss).

Fluorescent immunohistochemistry was used to assess Gli1 nuclear translocation. Cultured MG63 and MG63R cells were fixed and then incubated with primary antibody against Gli1 (Cell Signaling Technology, 1: 500) at 4°C for 8 h. Then, the secondary antibody conjugated with Alexa 488 (Invitrogen) and DAPI (Invitrogen) were used to incubate the cells. Quenching of fluorescence was alleviated using the SlowFade Light Antifade kit (Molecular Probes). Samples were excited at 519 nm and observed at 442 nm and 49 5nm with an inverse fluorescence microscope (Axio Imager 2, Zeiss). The fluorescent intensity was determined by using Zeiss Physiology software (Ver. 3.2, Zeiss).