Hy 17471a

Cells were treated with inhibitor (or solvent control) for one hour at the specified concentrations in the appropriate medium prior to infection (pretreatment). Pretreatment was removed and cells were subsequently infected as described. Fresh medium containing inhibitor (or solvent control) was reapplied. HA15 (MedChemExpress, HY-100437, Monmouth Junction, NJ, USA), VER-155008 (MedChemExpress, HY-10941, Monmouth Junction, NJ, USA), BTB06584 (MedChemExpress, HY-15877, Monmouth Junction, NJ, USA), and Enterostatin (Abbiotec, 350165, Escondido, CA, USA) were dissolved in DMSO to a final concentration of 50mM. Metformin (MedChemExpress, HY-17471A, Monmouth Junction, NJ, USA) and Fluvastatin (MedChemExpress, HY-14664A, Monmouth Junction, NJ, USA) were dissolved in water to a final concentration of 100 mM.

A wound healing assay was used to evaluate cell migration. Briefly, cells were seeded and cultured to reach confluence, and then a wound was generated using a pipette tip. Mitomycin-C (5 mg/ml, purity: 99.74%, Selleck, S8146) was used to inhibit cell proliferation during this experiment. After an incubation of 0, 12, or 24 h, wound images were taken using a Model IX70 Microscope (Olympus, Tokyo, Japan). The width of wounds was measured and cell migration was calculated.
To observe the effect of metformin hydrochloride (MET, purity: 99.98%, MedChemExpress, HY-17471A) on cell migration, cells were pretreated with MET (100 μM) for 72 h, followed by cell migration analysis using wound healing assay as described above. The half-life of MET is 4–8.7 h (Dunn and Peters, 1995 (link)) and the media (containing glucose with or without MET) in this and other MET related experiments were changed every 24 h (Niu et al., 2019 (link)).

Ascending aortic tissues were taken from patients who underwent heart transplantation. Then, the tissues were placed into culture dishes filled with Dulbecco's modified Eagle's medium (DMEM)/F12 (SH30023.01; HyClone) at 4°C. Intimal and residual adventitial tissues were stripped under a stereomicroscope. Subsequently, the middle layer of the aortic wall was cut into small pieces (1–2 mm) and transferred to cell culture flasks with 5 ml of DMEM/F12 supplemented with 10% fetal bovine serum (SH30084.03; HyClone) and 1% penicillin-streptomycin (15140-122; ThermoFisher Scientific). A few days later, long, spindle-shaped HASMCs were observed and then passaged when the degree of cell fusion reached approximately 80%. Human aortic smooth muscle cells were cultured at 37°C in a humidified incubator with 5% CO₂. Human aortic smooth muscle cells were infected with lentivirus containing shLOXL2 or shLOXL3 to knockdown LOXL2 or LOXL3 according to our previously reported methods (3 (link), 17 (link)). Human aortic smooth muscle cells were starved for 12 h and then treated with losartan (10 and 100 μM, S1359; Selleck) and metformin (5 and 10 mM, HY17471A; MedChemExpress) for 48 h, with DMSO treatment serving as a control.