Myocardin

Freshly dissected tissues were fixed and prepared for histological examinations. H&E and Masson’s trichrome staining as well as immunochemistry (IHC) were performed according to the manufacturer’s instructions. Sections were cut at 4 μm and incubated with myocardin (1:100, Abcam, UK) and α-SMA (1:50, Santa Cruz, USA). Digital images were acquired with an Olympus microscope (Olympus, Shinjuku Monolith, Japan), and the SM-to-collagen ratio of Masson’s trichrome staining was evaluated using Image-Pro Plus 6.0.

Different groups of ASC cells were seeded and grown on glass coverslips before fixation with 4% paraformaldehyde and were incubated with 0.25% Triton X-100/1% bovine serum albumin (BSA)/PBS. The primary antibodies, α-SMA (1:50, Santa Cruz, USA), calponin (1:100, Santa Cruz, USA), myocardin (1:100, Abcam, UK), and SRF (1:50, CST, USA), were incubated at 4 °C overnight, followed by double staining of secondary antibodies (all 1:50, Bioworld, USA) with FITC (green) or TRITC (red) at room temperature and protected from light for 1 h. 4′,6-Diamidino-2-phenylindole (DAPI, Abcam, UK) was introduced to locate the cell nucleus. The contractile proteins α-SMA and calponin and the molecular colocalization of myocardin and SRF were detected by an Olympus laser scanning confocal microscope (Olympus, Shinjuku Monolith, Japan).

Briefly, protein was extracted from the aorta using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) and quantified using a BCA Protein Kit (CWBio, China). Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 1% bovine serum albumin (BSA) for 1 h, and then incubated with primary antibodies against p-AKT (Cell Signaling Technology, USA), AKT (Cell Signaling Technology, USA), p-mTOR (Cell Signaling Technology, USA), mTOR (Cell Signaling Technology, USA), p62 (Cell Signaling Technology, USA), LC3 (Sigma-Aldrich, USA), SM22α (Abcam, UK), α-SMA (Abcam, UK), myocardin (Abcam, UK), OPN (Abcam, UK), matrix metalloproteinase-2 (MMP-2; Novus, USA), matrix metalloproteinase-9 (MMP-9; Abcam, UK), p65 (Cell Signaling Technology, USA), TNF-α (Abcam, UK), and β-actin (Abcam, UK), overnight at 4°C. Next, membranes were washed three times with tris buffered saline tween (TBST) and incubated for 1 h with secondary antibodies (Abcam, UK). Proteins were detected using enhanced chemiluminescent reagents (Thermo Fisher Scientific, USA) and quantified using ImageJ software (NIH, USA).