Oil red o

The mouse heart and aorta were perfused, dissected, and cleaned from surrounding fat tissue. The aorta was fixed with 4% PFA, opened longitudinally, pinned flat onto the plate, and stained with Oil Red O (Beyotime, Shanghai, China). The percentage of the area of red stained lipids with Oil Red O to the total area is calculated to analyze lipid deposition. The mouse hearts and the vascular strips were immersed in 20% sucrose solution for 24 h, embedded in OCT, and then cut into tissue sections (serial sections, 6 μm). The serial sections were cut from the onset of the aortic valves and then stained with Oil Red O staining. The quantification of the atherosclerotic lesion area in the full-length aorta and aortic root lesion area stained with Oil Red O was quantitated using the Image J software. The mouse liver, spleen, and kidney were collected and fixed with 4% PFA. After being immersed in 20% sucrose solution for 24 h, they were embedded in OCT and cut into 6 μm tissue sections. Sections were stained with hematoxylin-eosin staining.

After lineage differentiation, cells were washed twice with PBS and fixed with 4 % formalin for 10 min at room temperature. For Oil red O staining, fixed cells were then stained with Oil red O reagent (Beyotime) for 1 h at room temperature; for Alizarin red staining, fixed cells were stained with 1% Alizarin red (Leagene, Beijing, China) with pH 4.2 for 30 min at room temperature; for Alcian Blue staining, fixed cells were then colored for 30 min in a 1% Alcian Blue solution (OriCell). Finally, cells were washed with water for three times to remove unbound dye and photographed under light microscopy.

To induce excessive lipid accumulation in an in vitro NAFLD cell model, HepG2 cells were seeded at 5 × 10⁴ cells per well in a 96-wells plate, and divided into five groups: blank, FFA induction, and SPE treatment group (1, 10, and 20 μg/ml). FFA (1 mM) was prepared by dissolving oleic acid and palmitic acid (concentration ratio 2:1). HepG2 cells were firstly incubated with SPE solution at 37°C for 4 h, flowing with the incubation of FFA–BSA complex at 37°C for 24 h. Control cells were treated with 1% BSA only. HepG2 cells were washed with PBS, fixed in 4% paraformaldehyde for 10 min, and then stained with Oil Red O at room temperature (Beyotime, Shanghai, China) for 30 min. The stained cells were washed with distilled water before photographing. For lipid content quantification, the stained cells were incubated with isopropanol for 10 min at room temperature, and the absorbance at 500 nm was measured. The results were expressed as percentage of viable cells with respect to untreated control cells. To determine the synthesis of triglyceride, cells were seeded in 6-well plates, treated under the same conditions, harvested with absolute ethyl alcohol, and fully lysed by ultrasonicator treatment. After centrifugation, the supernatant was collected to measure the concentration of triglyceride according to the standard samples.

The 3T3-L1 cells were planted in a 24-well culture growth (pyruvate-free) DMEM (Gibco, Camarillo, CA, USA) medium supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beit-Haemek, Israel) and penicillin–streptomycin (100 U/mL penicillin, 100 mg/L streptomycin) at 37 °C in a humidified atmosphere (5% CO₂, 95% air). 3T3-L1 cells were transfected with pcDNA3.1(+)-GALNT5 overexpression plasmid or siRNA when grown to 70~80% confluency using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. RT-qPCR and Oil Red O (ORO) staining were performed on the transfected cells. Lipid droplet generation of 3T3-L1 cells and sections were visualized using Oil Red O (Beyotime, Shanghai, China) staining.