P62 sqstm1

The cultured cells were solubilized in lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris–HCl, 5 mmol/L EDTA–2Na, 1% Triton X-100, and 1 tablet/10 mL complete mini EDTA-free) and centrifuged at 15,000 × g at 4 °C for 30 min. Samples were separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were first blocked in a buffer containing 25 mmol/L Tris–HCl (pH 7.4), 150 mmol/L NaCl, 0.1% Tween 20, and 4% skim milk for 1 h and then incubated with primary antibodies at 4 °C overnight. This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h. Primary antibodies against human p21^Waf1/Cip1, p16^INK4a, phosphorylated H2AX (Ser139, γ-H2AX), retinoblastoma (Rb), phosphorylated Rb (Ser780, pRb), cyclin D, and caspase-3 were all purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies against human cyclin A (Novocastra Laboratories Ltd., Newcastle, UK), p16^INK4a (BD Biosciences, Inc., Farmingdale, NY, USA), Ki-67 (Dako from Agilent, Santa Clara, CA, USA), p62/SQSTM1, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were also used. The immunoreactive proteins were then detected by enhanced chemiluminescence (GE Healthcare, Fairfield, CT, USA). Immunoblots were quantified using the CS Analyzer 3.0 software (ATTO, Tokyo); β-actin expression was used as the internal control.

The vessels were fixed and embedded in paraffin (Sigma-Aldrich, USA) or frozen tissue matrix (OCT, Leica biosystems, Germany) for sections. Histological stainings for H&E, elastin stain (Sigma-Aldrich, USA), and Verhoeff’s Van Gieson stain were performed to assess the changes of vascular structure for neointima formation and failure of vein graft. The TUNEL (Roche, Switzerland), immunohistochemical (IHC), and immunofluorescent (IF) stainings were used to determine cell apoptosis and inflammatory responses using specific antibodies against inflammation markers, such as COX-2 (Abcam, UK). Autophagy formation was measured by puncta formation of LC3 (Santa Cruz, USA) and expression patterns of p62/SQSTM1, Beclin 1, Bcl-2, and Bcl-X(L) (Santa Cruz, USA). The CD31 antibody (R&D system) was used to identify the endothelial cells. The ultrastructure of autophagosome was observed by typical double membrane morphologies using a TEM (JEM-1400, JEOL, Japan).