Light microscope bx53 system

The porcine model of restenosis after stenting was generated as our previously described.²², ²³ After porcine were sacrificed, the coronary arteries were removed from the heart and fixed with 10% formalin, then paraffin embedded and sectioned into 5 μm. The sections were then stained with haematoxylin–eosin, and observed under the microscope. The expression and localization of METTL3 were determined according to a previously described immunofluorescence method.³, ⁴ Slides with tissue sections were dewaxed with paraformaldehyde and gradually hydrated with ethanol. Then, the slides were placed in EDTA repair solution at 100°C for 20 min. After the slides were blocked with blocking buffer (5% bovine serum albumin) at 37°C for 30 min, the primary antibody METTL3 (Proteintech 15,073‐1‐AP, 1:200 dilution) and α‐SMA (Abcam, ab7817, 1:200 dilution) were incubated for overnight at 4°C. Subsequently, the secondary antibodies (Alexa Fluor 568 donkey anti‐Rabbit IgG [H + L; Thermo Fisher Scientific, A10042] for METTL3 and the Alexa Fluor 488 donkey anti‐mouse IgG [H + L; Thermo Fisher Scientific, A21202] for α‐SMA) were incubated for 60 min at 37°C and followed with diamidino‐phenyl‐indole (DAPI) staining in the dark. After washing with PBS, an Olympus light microscope BX53 system was applied for images capture.

The human aorta sample was obtained after operation, and the samples were quickly put in 10% formalin for fixation after being washed with cold saline solution. After 3 days of fixation, the tissue specimens were dehydrated and embedded in paraffin by following standard histological procedures. Subsequently, sectioning was performed and slices were stained with H&E for morphological examination as previously described^{44 (link),45 (link)}. EVG staining was performed for elastic fiber assessment. The images were captured by using an Olympus light microscope BX53 system.

A Cell-Light™ Edu Apollo 567 In Vitro kit (C10310-1, RiboBio) was used to perform the EdU incorporation assay. HASMCs treated with DMSO or 0.5 μM JIB-04 were plated in 24-well plates at 2 × 10⁴ cells per well and incubated with EdU medium (50 µmol/L) made up of EdU solution and complete medium at a ratio of 1:1000 for 4 h. Cells were fixed with 4% paraformaldehyde for 30 min after washing with by PBS. Then, the cells were incubated with glycine (2 mg/mL) to neutralize paraformaldehyde and were incubated with a penetrant made up of PBS and 0.5% Triton X-100 for 10 min. Cells were colored with 1 × Apollo staining solution and then DNA was stained with DAPI after the incubation with 0.5% Triton X-100 PBS solution again. The cells were washed with PBS and observed by fluorescence microscopy. Images were captured by an Olympus light microscope BX53 system.

Immunofluorescence staining was performed as previously described 8 (link). The RAVSMCs and primary HAVSMCs were stained with α-SMA (ab7817, Abcam) antibody, and images were taken using an Olympus light microscope BX53 system.
The RAVSMCs were treated with 5 μM BIX01294 for 24 h, and then fixed with 2.5% cold glutaraldehyde for 30 min after washing with PBS twice. Transmission electron microscope (TEM) analysis was conducted as previously reported 8 (link).