Nucleolin

HUVECs were plated in 60-mm culture dishes (6 × 10⁵ cells/dish), incubated with LPS (0.1 µg/mL) for 4 h, and then incubated with DMVR-2 (25 µg/mL) for 3 h and 6 h and the respective controls. The cells were treated with trypsin and washed twice with PBS. Subsequently, the cell pellet was subjected to cell lysis and separation of the cytoplasmic and nuclear protein extraction using a NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific). The protein concentration was determined by a Bradford-Solution for Protein Determination (Applichem, Monza, Italy) using BSA as a standard. The proteins were fractionated on SDS-PAGE and transferred into nitrocellulose membranes. After electro transferring, the nitrocellulose membrane was blocked with 10% nonfat dry milk for 1 h at room temperature and then immunoblotted with appropriate primary antibodies, against Nf-kB p65 (F-6) (Santa Cruz Biotechnology), GAPDH (Santa Cruz Biotechnology) and Nucleolin (Sigma-Aldrich), O/N at 4 °C. The signals were visualized with the appropriate horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence (Amersham Biosciences-GE Healthcare, NY, USA). Densitometric analyses were carried out using the ImageJ software Java8. The experiments were performed in duplicate.

For the aptamer-specific assembly, 500 nM DNA tiles were mixed with 1.5 μM nucleolin (Sigma Aldrich, cat. no. N2662) in 1× TAE buffer containing 20 mM MgCl₂. For subsequent disassembly of the DNA filaments, 10 mM ATP was added to the solution. In the case of the strand-displacement-mediated (de-)polymerization, 500 nM DNA tiles were mixed with 10 μM invader strands and encapsulated into droplets immediately afterwards, which induced the disassembly of the DNA filaments. By addition of 37.5 μM anti-invader strands directly before encapsulation, the filaments were reassembled.

Following various treatments, VSMCs cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Shanghai Biyuntian Biotechnology, Ltd.). The protein concentration was determined using BCA assay. Whole‐cell lysates were subjected to SDS‐PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes. The blots were incubated with respective primary antibodies against nucleolin (rabbit polyclonal antibody, Sigma), α‐SM‐actin (mouse monoclonal antibody, Boster Biotech), SM22a (rabbit polyclonal antibody, Abcam), calponin (mouse monoclonal antibody, Abcam), OPN, EGF, PDGF‐BB (rabbit polyclonal antibody, Abcam) and β‐actin (mouse monoclonal antibody, Abcam) at 25°C for 2 hours. Subsequently, the blots were incubated with peroxidase‐conjugated secondary antibodies at 25°C for 1 hours. Immunoreactive bands were visualized utilizing enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions, and the densitometry analysis was performed by scion image software.