Integrin α5

Cells were collected and lysed in cold RIPA buffer, and the protein concentration was determined using a bicinchoninic acid assay kit (Pierce Biotechnology, Waltham, MA, USA). Protein samples were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (PVDF, Merck Millipore). The PVDF membrane was blocked in 5% nonfat milk buffer and then incubated with primary antibodies against E-cadherin (1:500, BD Biosciences), fibronectin (1:500, BioWorld), Pdx-1 (1:500, Abcam), Ki67 (1:500, Santa Cruz), extracellular signal-regulated kinase (ERK) 1/2 (1:500, Abcam), p-ERK1/2 (1:500, Cell Signaling Technology, Danvers, MA, USA), focal adhesion kinase (FAK, 1:500, Cell Signaling Technology), p-FAK (1:500, Cell Signaling Technology), Akt (1:500, Cell Signaling Technology), p-Akt (1:500, Cell Signaling Technology), integrin α5 (1:500, Abcam), integrin β1 (1:500, Abcam), cyclin D1 (1:500, Santa Cruz), p27 (1:500, Cell Signaling Technology), and β-actin (1:1,000, Santa Cruz) overnight at 4°C. The PVDF membrane was washed in PBST and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000, Santa Cruz) at 37°C for 1 hour. The target protein band on the PVDF membrane was observed using an electrochemiluminescence kit (Pierce).

For evaluation of the protein levels of α₅ and α_v integrins and NaBC1 transporter, we used In-Cell Western quantification. 10.000 cells cm⁻² of C2C12 cells were seeded onto FN-coated substrates during 1.5 h at 37 °C and 5% CO₂. Cells were then fixed using fixative buffer (10 mL)formaldehyde, 90 mL PBS, 2 g sucrose) at 37 °C for 15 min and then permeabilized in cold methanol at 40 °C for 5 min. Cells were then blocked in 0.5% blocking buffer (non-fat dry milk powder in 0.1% PBST buffer) at RT for 2 h followed by 3 washes of 10 min with 0.1% PBST. Cells were then incubated with primary antibodies: NaBC1 (abcam, 1:200), integrin α₅ (abcam, 1:500) and anti-integrin α_v (abcam, 1:500) diluted in blocking buffer at 4 °C overnight. After 3 washes of 10 min with 0.1% PBST buffer, cells were incubated with 1:800 diluted infrared-labeled secondary antibody IRDye 800CW (LI-COR) and 1:500 diluted CellTag 700 Stain (LI-COR) at RT for 1 h, followed by 5 washes of 10 min with 0.1% PBST. Samples were then dried overnight at room temperature. Infrared signal was detected using an Odyssey infrared imaging system. Four biological replicas were analyzed.

After fixation and rehydration of 6 µm sections from the femoral artery, the slides were pre-incubated with 10% normal goat serum (Zymed® Laboratories Inc., San Francisco, CA, USA) and then incubated with antibodies against α-SMA (Sigma-Aldrich, Munich, Germany), CD31 (BD Pharmingen, Franklin Lakes, NJ, USA), CD45 (BD Pharmingen), CD68 (Serotec, Oxford, UK), Ki-67 (Abcam, Cambridge, UK), Integrin α5 (Abcam), or Sirt1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Ensuing incubations were carried out with Cy5- or Cy3-coupled secondary antibodies (Molecular Probes, Eugene, OR, USA) and counterstained with nuclear 4.6-diamidino-2-phenylindole (DAPI) (Linaris, Wertheim, Germany). Monoclonal antibodies to α-SMA were labelled directly with Cy3. Negative controls were conducted by substituting the primary antibody with an appropriate species- and isotype-matched control antibody (Santa Cruz Biotechnology). The number of apoptotic SMC was quantified by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) according to the supplier's instructions (in situ cell death detection kit, Roche).

Protein samples were separated in NuPAGE 4–12% Bis-Tris protein gels (#NP0336BOX; ThermoFisher Scientific) and transferred to polyvinylidene fluoride membrane (#LC2002; ThermoFisher Scientific) for antibody probing. The blots were blocked with 5% blocker (#1706406; Bio-Rad, Hercules, CA) in Tris-buffer saline containing 0.05% Tween-20. Blots were then incubated with primary antibodies overnight at 4°C before incubating with horseradish peroxidase–conjugated secondary antibodies for 1 h at room temperature. The following antibodies were used in Western blotting: Ror2 (1:1000; Developmental Studies Hybridoma Bank), integrin α₅ (1:1000; #ab150361; Abcam), fibronectin 1 (1:1000; #610077; BD Biosciences), FAK (1:1000; #3285; CST), phospho-FAK (1:1000; #44-626G; ThermoFisher Scientific), ROCK1 (1:1000; #4035; CST), RhoA (1:1000; #2117; CST), cofilin 1 (1:1000; #5175; CST), MLCK (1:1000; #M7905; Sigma-Aldrich), total MLCII (1:1000; #3672; CST), p-MLCII^Thr18/Ser19 (1:1000; #3674; CST), p-MLCII^Ser19 (1:1000; #3671; CST), and GAPDH (1:2500; #5174; CST).