Caldesmon

Frozen tissues or cells were lysed with RIPA lysis buffer containing PMSF, protease and phosphatase inhibitors (Roche Diagnostics). Protein concentration was quantified using a BCA protein assay kit (Pierce). Samples containing 30 μg of protein were separated by SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% fat-free milk before incubation with primary antibodies specifically recognizing PKR (ab32052, 1:1000, Abcam), p-PKR (ab32036, 1:1000, Abcam), human GSDMD (ab210070, 1:1000, Abcam), rabbit anti-human N-GSDMD antibody (EPR20829-408) (ab215203, 1:1000, Abcam) and IL-1β (MAB201, 1:1000, R&D system), α-SMA (1:1000, Proteintech), SM22α (1:1000, Proteintech), calponin (1:1000, Proteintech), caldesmon (1:1000, Proteintech), thrombospondin (1:1000, Proteintech) and osteopontin (1:1000, Proteintech). Immunoreactivity was detected by an enhanced chemiluminescence system, and bands were scanned with a Bio-Rad Imager. Individual band intensity was determined with ImageJ software.

Sections were fixed in 4% formalin for 48h and then dehydrated, embedded in paraffin and cut into 4μm sections. Morphological changes of aortic walls were observed in sections stained with Hematoxylin-eosin staining. The collagen deposition and elastin content of aortic walls was evaluated with Masson trichrome staining. The media thickness, relative positive staining area for collagen and elastin were measured by an image analysis system (Image-ProPlus, Version 6.0; Media Cybernetics). Immunohistochemistry of tissue sections were performed with the following primary antibodies: anti-PKR (1:100, Abcam), anti-p-PKR (1:100, Abcam), anti-α-SMA (1:100, Proteintech), CD31 (1:100, Abcam), SM22α (1:100, Proteintech), Calponin (1:100, Proteintech), Caldesmon (1:100, Proteintech), Osteopontin (1:100, Proteintech), Thrombospondin (1:100, Proteintech), p53 (1:100, Proteintech), p16^INK4a (1:100, Proteintech).