Sc 130617

Immunofluorescence staining was performed on 4 μm paraffin cross-sections from the carotid arteries. VSMCs were prepared as described above. The sections were deparaffinized with xylene and rehydrated, cells were fixed by 4% paraformaldehyde and then were permeabilized by incubation with 0.1% Triton X-100 in PBS. Non-specific sites were blocked by incubation in 10% normal goat serum (710027, KPL, USA) for 1 h. Then the sections or cells were incubated with primary antibodies at 4 °C overnight. The primary antibodies were anti-SMα-actin (mouse monoclonal 1:100, sc-130617, Santa Cruz), VCAM-1 (rabbit monoclonal, 1:100, ab134047, Abcam), anti-OTUD7B (rabbit polyclonal, 1:50, 16605-1-AP, Proteintech), anti-NMHC IIA (rabbit polyclonal, 1:50, 11128-1-AP, Proteintech), anti-NMHC IIA (mouse monoclonal, 1:50, 60233-1-Ig, Proteintech). Secondary antibodies were rhodamine-labeled antibody to rabbit IgG (1:50, 031506, KPL, USA) and fluorescein-labeled antibody to mouse IgG (1:50, 021815, KPL, USA), or rhodamine-labeled antibody to mouse IgG (1:50, 031806, KPL, USA) and fluorescein-labeled antibody to rabbit IgG (1:50, 021516, KPL, USA). Nuclei were stained with DAPI (1:50, 157574, MB biomedical) in each experiment. Images were captured by confocal microscopy (DM6000 CFS, Leica) and processed by LAS AF software.

Piceatannol was purchased from Future Chem (Seoul, Korea). Anti-alpha smooth muscle actin (α-SMA; 1:1000, sc-130617), anti-CTGF (1:1000, sc-14939), anti-HDAC3 (1:1000, sc-11417), anti-HDAC4 (1:1000, sc-11418), anti-HDAC5 (1:1000, sc-133225), anti-TGF-β1 (1:1000, sc-146), anti-JNK (1:1000, sc-7345), anti-ERK1 (1:1000, sc-271269), and anti-GAPDH (1:1000, sc-32233) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Primary antibodies against collagen type I (1:1000, ab34710), HDAC2 (1:1000, ab12169), HDAC8 (1:1000, ab137474), and HDAC10 (1:1000, ab53096) were purchased from Abcam (Cambridge, MA, USA). Anti-fibronectin antibody (1:1000, MA5-11981) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-HDAC1 antibody (1:1000, 06–720) was purchased from Merck Millipore (Darmstadt, Germany). Anti-HDAC6 (1:1000, 7612), anti-Smad3 (1:1000, 9523), anti-Smad2 (1:1000, 3103), anti-Smad4 (1:1000, 9515), anti-p-Smad3 (1:1000, 9520), anti-p-JNK (1:1000, 9251), anti-p-p38 (1:1000, 4511), anti-p-ERK1/2 (1:1000, 4370), and anti-p38 (1:1000, 8690) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

The injured carotid arteries by ligation were anatomically localized and cut as described previously 23 (link). In brief, the common carotid arteries were approximately 9 mm long, of which the proximal and distal 2 mm were discarded and the remaining portion (approximately 5 mm) was cut in half. The two segments were embedded in paraffin, and serial sections (4 μm thick) were cut for analysis by immunofluorescent staining for SM α-actin (mouse monoclonal, 1:50, sc-130617, Santa Cruz), OTUD7B (rabbit polyclonal, 1:50, 16605-1-AP, Proteintech) or VCAM-1 (rabbit monoclonal, 1:250, ab134047, Abcam) as well as by hematoxylin-eosin or Elastic-van Gieson staining for morphometry. Five or more sections spanning most of the vessel segment were analyzed for morphometry. The neointimal area and intima-to-media ratio were calculated using Image-Pro Plus Analyzer (version 5.1) software (Media Cybernetics, Silver Spring, MD) in a blinded manner.