Plate reader at 450 nm

Levels of total cholesterol (TC), triglycerides (TG), and high density lipoprotein-cholesterol (HDL-C) were measured using enzymatic assay kits in the UCLA Cardiovascular Core Facility. VLDL-C (very low density lipoprotein-cholesterol) and LDL-C (low density lipoprotein-cholesterol) were calculated based on the Friedewald equation^{56 (link),57 (link)}. The serum level of TNF-α, IL-1β and IL-6 were measured by Enzyme-linked immunosorbent assay (ELISA) using Ready-SET-go kits (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s protocol. The color reaction was stopped with the addition of Stop solution (BioLegend, San Diego, CA), and absorbance was read immediately using a plate reader at 450 nm (Bio-Rad Laboratories, Hercules, CA). The standard curve was calculated by plotting the standards against the absorbance values, and the cytokine levels were measured in pg/ml.

The cell proliferation ability was determined by CCK8, EdU and colony formation assays. For CCK-8 assay, Cell Counting Kit-8 was bought from DingGuo (Beijing, China), and cells were seeded into 96-well plates (5000 cells/well) with complete growth medium. After incubation for 24, 48, 72, and 96 h, respectively, 10 μL CCK-8 was added into each well, and then the cells were cultured for an additional 2 h. The absorbance value was tested by a plate reader at 450 nm (Bio-Rad, Hercules, CA, USA). 1 × 10⁵ cells were put into 24-well plates for EdU incorporation with an EdU detection kit (Ribobio, Guangzhou, China). The percentage of EdU-positive cells was quantified from four random fields per well. For colony formation assays, 2.5 × 10² cells, were plated onto six-well plates and incubated for 14 days, fixed with 4% paraform, then stained with 0.1% crystal violet, and the number of colonies in four random fields were counted under an inverted microscope. The experiments were performed in triplicate.