Sab4200539

Whole-cell proteins were extracted and subjected to standard western blot analysis as described previously [41 (link)]. Antibodies against MYOCD (SAB4200539, Sigma, Massachusetts, USA), ACTA2(ab7817, Abcam, Massachusetts, USA), SM22α (ab14106, Abcam, Massachusetts, USA), PCNA (sc-56, Santa Cruz, California, United States), OPN (sc21742, Santa Cruz, California, United States), LC3 (NB100-2220, Novus Biologicals, Colorado, USA), P62 (610833,BD Transduction Laboratories, USA), Beclin1 (ab207612, Abcam, Massachusetts, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, Abcam, Massachusetts, USA) were used to probe for the target proteins. Secondary antibodies, including IRDye^®800CW anti-rabbit secondary antibody and IRDye^®680 anti-mouse secondary antibody, were purchased from Li-COR (Lincoln, Michigan, USA). Signals were detected by an Odyssey™ Infrared Imaging System (LI-COR, Michigan, USA), and the bands were quantified by Image-Pro Plus 5.1 software (MEDIA CYBERNETICS, Maryland, USA).

Aortic and cardiac tissues were fixed for 24 h in 4% formalin solution (BDH Prolabo), and subsequently dehydrated in 60% isopropanol (BDH Prolabo), followed by paraffin‐embedding. Aortic media thickness was measured on orcein‐stained sections of the aorta, which allows for accurate assessment of the inner and outer border of the media layer. Orcein staining was further used to assess elastin content and for counting elastin breaks and elastic laminae. The latter is assessed as the average of 8 measurements across the aortic wall. VSMC phenotype was assessed using immunofluorescent myocardin staining (Sigma, sab4200539), and immunohistochemical staining for α‐smooth muscle actin (α‐SMA, Sigma, F‐3777) and proliferating cell nuclear antigen (PCNA, Biorad, MCA1558F). Cardiac fibrosis was assessed using trichrome Masson staining and cardiac hypertrophy was quantified on the cellular level using an anti‐laminin (Novus Biologicals, nb300‐144) staining. Five images off each mouse were recorded for this measurement in different cross‐sectional regions of the heart, and cross‐sectional area of 20 cardiomyocytes was measured per image (final average of 100 measurements). Microscopic images were acquired with Universal Grap 6.1 software using an Olympus BX4 microscope or Celena S fluorescence microscope and quantified using ImageJ software.