Ascorbate

SD rats aged 5–8 weeks were anesthetized on the sterile operating table, and the whole thoracic aorta was quickly isolated. Subsequently, the blood vessels were cut into tissue blocks of 1–2 mm size and soaked in T25 bottles (containing 2 mL fetal bovine serum) 30 min. The tissue blocks were placed at the bottom of the DMEM (ThermoFisher, USA) culture flask (containing 15% FBS) and cultured at 37°C in a 5% CO₂ incubator for 12 h. After a week, a large number of cells distributed around the tissue mass can be subcultured. The calcifying medium contained 10 mM β-glycerophosphate (β-GP, Beyotime, ST637) and 50 μg/mL ascorbate (Merck, USA, 134-03-2). The obtained VSMC was intervened differently, including control (5.5 mM glucose), high glucose (HG, 25 mM glucose), LIRA (100 nM), HG + LIRA (20 mM), HG + LY294002 (PI3K antagonist, 50 μM, MCE, USA, HY-10108), HG + LY294002 + LIRA, HG + PD98059 (ERK antagonist, 50 μM, MCE, USA, HY-12028), HG + PD98059 + LIRA, and HG + Exe (9-39) (GLP-1 receptor antagonist, 200 nM, MCE, USA, HY-P0264) + LIRA.

To perform SiriusRed assay, MRC-5 cells were seeded (n = 6 well/treatment group) into 96-well tissue culture plates at a density of 10⁴ cells/well. After 24 h of plating, cells were treated for 48 h with recombinant TGF-ß in the absence or presence of nintedanib diluted in culture medium containing 0.1% FBS and 100 μM ascorbate (Merck).
The collagen deposition was determined based on a basic histological dye SiriusRed, incorporating into the triple helical collagen molecules [50 (link)]. After removing supernatants, cells were incubated in a fixative solution containing 26% EtOH, 3.7% formaldehyde, 2% glacial acetic acid for 15 min at room temperature. Samples were stained for 1 h at room temperature with 0.1% solution of SiriusRed (DirectRed80) dissolved in 1% acetic acid, then washed three times with 200 μL of 0.1 M HCl, and finally the bounded dye was dissolved by adding 100 μL of 0.1 M NaOH (all reagents were purchased from Merck). Absorbance was recorded at 544 nm and at 690 nm as background in a SPECTROstar Nano microplate reader using SPECTROstar Nano MARS v3.32 software.

The following reagents were purchased from Sigma Aldrich (St. Louis, MO): antimycin A, ADP, ascorbate, azide, FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone), malate, oligomycin, pyruvate, rotenone, succinate, and TMPD (N,N,N′,N′-Tetramethyl-p-phenylenediamine). All other reagents purchased are noted elsewhere.

After metabolic labeling by AHA, a click reaction was performed as previously published (Dieterich et al., 2007 (link)). Briefly, a cocktail of 500 μM alkyne-biotin reagent (Sigma-Aldrich), 1 mM Copper Sulfate (CuSO_4, Sigma-Aldrich), 6 mM 3-(4-((bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)propanol (BTTP, Click Chemistry Tools) and 6 mM Ascorbate (Sigma-Aldrich) was added to 500 μg cell lysate. The samples were incubated for 3 hours at room temperature.

Osteogenic differentiation was conducted. In brief, ADSCs at passage 2 were induced by 2.5 weeks of feeding (twice a week) with osteogenic induction medium consisting of 100 nM dexamethasone, 10 mM β-glycerophosphate, 0.2 mM ascorbate (all from Sigma-Aldrich Chemical Company, St Louis, MO, USA), and 10% fetal calf serum (FCS) in DMEM/F12 basal medium. Osteogenic differentiation was subsequently confirmed by mineralized matrix deposition by 0.2% alizarin red-S staining.
Adipogenic differentiation was then performed. In short, the cells were induced by 3 cycles of induction/maintenance using adipogenic induction medium consisting of 1 mM dexamethasone, 0.5 mM 3-isobutyl-1-methyl-xanthine (IBMX), 10 μg/ml recombinant human insulin, 100 mM indomethacin (all from Sigma-Aldrich Chemical Company, St Louis, MO, USA), and 10% FCS, and using adipogenic maintenance medium comprising of only 10 μg/ml recombinant human insulin and 10% FCS. After that, the induced cells were subjected to incubation for another 7 days in adipogenic maintenance medium. Adipogenic differentiation was then confirmed by the formation of neutral lipid-vacuoles stainable with 0.18% oil Red-O for 5 min (Sigma-Aldrich Chemical Company, St Louis, MO, USA). Primary normal human dermal fibroblasts served as negative control (NC). Each experiment was run in triplicate.