Alizarin red staining

A schematic diagram of the experimental procedure is shown in Figure 1. In this experiment, hAMSCs were isolated from the amnions of human placentas in accordance with a previous protocol, and relevant informed consent was provided by each donor before the operation.^{35 (link)-37 (link)}
LG-DMEM/F12 medium containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% L-glutamine, and 1% nonessential amino acids was used to culture the hAMSCs. The morphology of the hAMSCs was observed under an inverted microscope after three days of cultivation. Third-generation (P3) hAMSCs were used in subsequent experiments. The stemness of the hAMSCs was tested by osteogenic and chondrogenic differentiation according to previous studies.^{35 (link)-38 (link)}
Briefly, hAMSCs were seeded in a six-well plate, and the osteogenic medium (10% FBS, 50 mg/ml ascorbate, 100 nM dexamethasone, and 10 mM β-glycerophosphate) was added when the cell density reached 60%. The results were detected with the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China) and alizarin red staining (Solarbio, China). In terms of chondrogenic differentiation, we used Chondrogenic Differentiation Basal Medium (Cyagen, USA) to induce hAMSCs for 14 days; alcian blue staining was used for detection (Solarbio).

Equal numbers of cultured cells from Hif-2α^fl/fl and Prx1-Cre;Hif-2α^fl/fl mice were replated to ensure that there was no difference in the density of the cells of the different genotypes. On the second day of culture, the medium was replaced with adipogenic (7 d) or osteogenic (7 d or 14 d) differentiation medium. The osteogenic differentiation medium consisted of MEM supplemented with 10 mmol/L β-glycerol phosphate disodium salt, 10 nM DEX (D1756) and 50 μM L-ascorbic acid[19 (link)]. Fourteen days later, the percentage of colonies that contained osteoblasts was quantified by alizarin red staining (1%, pH = 4.2; Solarbio). Adipogenic differentiation was induced by the addition of α-MEM containing 10% fetal calf serum, insulin (1 μg/mL; Sigma), 1 μM DEX (Sigma), and 0.5 mmol/L 3-isobutyl-1-methylxanthine (Sigma). The differentiation of adipocytes was monitored by Oil red O staining[11 ].

Paraffin-embedded aortic valve samples were cut into 5-mm-thick sections and then incubated for 2 hours at 72°C before being deparaffinized with xylene and alcohol. H&E-stained sections were examined to identify the differences between noncalcified and calcified aortic valves. Immunohistochemistry detection of Piezo1 and GLS1 in mouse aortic valve leaflets was performed. For immunohistochemistry, after antigen retrieval was performed with a microwave, the prepared sections were incubated in 3% H₂O₂ for 10 min. Then, the sections were rinsed with PBS and blocked in 5% bovine serum albumin for 30 min at room temperature, followed by incubation with primary antibodies overnight at 4°C and HRP-conjugated secondary antibodies for 30 min at room temperature. Then, diaminobenzidine was used as a chromogen to visualize positive cells. The primary antibodies we used in immunohistochemistry staining were Piezo1 (1:100; Proteintech, 15939-1-AP, China) and GLS1 (1:100; Abmart, T55719, China). The secondary antibody was goat anti-rabbit HRP-conjugated antibody (1:200; FUDE Biological Technology, FDR007, China).
The deparaffinized and rehydrated sections were stained with von Kossa solution and alizarin red staining (Solarbio, China) to detect calcium deposits. Masson’s trichrome staining was performed to detect collagen deposition.