Gtx100554

The transfected cells on coverslips were fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were then permeabilized in 0.1% Triton X-100/PBS for 10 min, followed by 1 h of treatment with a blocking solution consisting of normal goat serum (1:200, Abcam, ab7481) in PBS. The pan-SOD1 antibody (1:400, Genetex; GTX100554) solution, diluted in the blocking solution, was added to the cell solution overnight at 4 °C. After washing with PBS, we incubated the cells with Alexa Fluor 488 antibody (Goat anti-Rabbit IgG, 1:500, Thermo Fisher Scientific, #A-11008) at 4 °C for 6 h. The nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI), and the endoplasmic reticulum (ER) was stained using ER-Tracker^TM Red dye (Invitrogen) for 10 min. After washing the coverslips three times with PBS, we mounted the coverslips with mounting solution (H-5501; Vector Laboratories) for fluorescence microscopy (Zeiss).
For inclusion positive cells counting, immunofluorescence images with SOD1 antibodies were counted in randomly selected fields. Depending on SOD1 staining, inclusion positive cells were counted based on the strong intensity of SOD1 and expressed as a percentage of total cells counted.

Cells and aorta tissue extracts (30 μg/mL of protein) were subjected to 10% SDS-PAGE gels and transferred to nitrocellulose by electroblotting. Primary antibodies against Runx2 (1:1000, sc-10758, Santa Cruz, Dallas, TX, USA), α-SMA (1:1000, #2547, Sigma-Aldrich, Saint Louis, MO, USA), SM22-α (1:1000, ab14106, Abcam, Cambridge, MA, USA), BMP-2 (1:1000, ARG57829, Arigo, Santa Cruz, Dallas, TX, USA), SOD1 (1;1000, GTX100554, GeneTex, Irvine, CA, USA), and SOD2 (1;1000, GTX116093, GeneTex, Irvine, CA, USA) were applied, followed by horseradish peroxidase (HRP)-linked secondary antibodies (Millipore, St. Louis, MO, USA) for 1 h at room temperature. Protein detection was visualized by chemiluminescence reagents (Millipore, St. Louis, MO, USA) and captured with a charged-couple device (CCD) camera (MiniChemi- Chemiluninescence, Sage Creation Sciences, BioRiver, Co., Ltd., Beijing, China). The immunoreactive bands were quantitatively determined using ImageJ software. Internal control was confirmed by β-actin (1:1000, sc-47778, Santa Cruz, Dallas, TX, USA).