P16ink4a

Cell lysates were prepared using RIPA buffer (9806, Cell Signaling) supplemented with protease and phosphatase inhibitors (11836153001 and 4906845001; Roche) according to manufacturer's instructions. Lysates were mixed with 4× Loading Buffer (928‐40004, Li‐Cor) and denatured at 95°C for 5 min prior to electrophoresis. SDS‐PAGE was performed with the NuPAGE® electrophoresis system (Thermo Fischer) including 4%–12% Bis‐Tris gels (Thermo Fischer, # NP0336BOX) and MES SDS Running Buffer (NP0002; Thermo Fisher) according to the manufacturer's instructions. Western blotting onto nitrocellulose membranes (10600002; GE Healthcare) was performed inside an XCell II™ Blot Module (Thermo Fischer) for 1 h at 30 V constant (25 mM Tris‐BASE; 192 mM Glycine, 20% (v/v) Methanol transfer buffer). Membranes were washed with TBS (15 mM Tris–HCl pH 7.6; 136 mM NaCl) and blocked with TBS Blocking Buffer (927‐50000, Li‐Cor). Primary antibodies were incubated at 4°C overnight according to manufacturer's instructions. The following antibodies were used: Phospho‐Histone H2A.X (Ser139) (2577, Cell Signaling), Cyclin D1 (2978, Cell Signaling), Lamin B1 (13435, Cell Signaling), p16^Ink4a (80772, Cell Signaling), p21^Cip1 (2948, Cell Signaling), GAPDH (2118, Cell Signaling), FLAG (740001, Thermo Fischer), pMLC (3675, Cell Signaling), and alpha‐SMA (ab5694, Abcam).

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 total aortic protein was extracted with lysis buffer. The DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, United States) was applied to measure the concentration of each sample. The protein abundance was detected with antibodies against Sirt-1 (cat. no. 2028, Cell Signaling Technology, Danvers, MA, United States), p16^INK4A (CDKN2A, cat. no. 10883-1-AP, Proteintech Group Inc., Rosemont, IL, United States), adiponectin receptor-1 (AdipR-1, ab70362), β-actin monoclonal antibody (1: 1000, AC-15, Sigma-Aldrich), and p21 (ab109199, both from Abcam, Cambridge, United Kingdom). The membranes then were incubated with the secondary antibodies. The protein contents calculated from western blots were normalized by loading β-actin.