Inorganic phosphate

To induce osteogenic differentiation, hMSCs were incubated in 6-well microplates in DMEM low glucose with 10% FBS, 50 μg/mL ascorbic acid (Sigma-Aldrich), 10⁻⁷ M dexamethasone, 1% ATB-ATM solution and 3 mM inorganic phosphate (Sigma Aldrich). Medium was changed every two days. At day 21, cells were stained or frozen at -80°C until RNA isolation. For staining, cells were fixed in 4% PAF and stained in alizarin red solution (Sigma Aldrich) at pH 4.2.

For gene expression analysis, VICs were seeded in 12-well plates at 10,000 cells/cm² (Corning, New York, NY, USA). Cells were cultured in the absence or presence of inorganic phosphate (2.6 mM) (Sigma, St. Louis, MO, USA) and harvested after either 9 days or 24 h. VICs treated with either BMP2 (20 ng/mL) or TGF-β (10 ng/mL) (R&D Systems, Minneapolis, MN, USA) for 24 h were used for determination of PRG4 mRNA levels. The effects of PRG4 on BMP2, RUNX2, and SOX9 mRNA levels were determined after 24-h and 9-day incubation of VICs cultured in the absence or presence of hrPRG4 (10 and 100 μg/mL). At the end of the incubation periods, total VIC RNA was isolated as described above. Real-time PCR was performed on a 7900HT Fast RealTime PCR system (Perkin-Elmer Applied Biosystems, Norwalk, CT, USA) as previously described [19 (link)] using Taqman Assay-on-Demand (Thermo Fisher Scientific, Waltham, MA, USA) for PRG4 (Hs00981633_m1), BMP2 (Hs00154192_m1), RUNX2 (Hs00231692_m1). and SRY-Box transcription factor 9 (SOX9) (Hs00165814_m1). Relative mRNA expression of target genes was quantified by the 2-ΔCT method using hypoxanthine phosphoribosyltransferase 1 (HPRT) (Hs02800695_m1) as endogenous control.

Primary VSMCs were seeded in growth medium at a density of 1.5 × 10⁴/cm² in multi-well plates. Calcification was induced as previously described [16 (link)]. In brief, cells were grown to confluence (day 0) and switched to calcification medium, which was prepared by adding inorganic phosphate (a mixture of NaH₂PO₄ and Na₂HPO₄, pH 7.4) (Sigma-Aldrich), to reach a final concentration of 3 mM phosphate. VSMCs were incubated for up to 14 days in 95% air/5% CO₂ and medium was changed every third/fourth day. Recombinant mouse BMP-9, ALK1-Fc or 3 μM ALP inhibitor was added at day 0.

Primary VIC cultures were seeded at a density of 10⁴ cells·cm⁻² in culture medium with 5% FBS and stimulated with DMSO, nilotinib, imatinib, 7rh (all from Sigma Aldrich, St. Louis, MO, USA), or WRG‐28 (ProbeChem Biochemicals, Shaghai, China). VICs were cultured in polystyrene plates (TPP) for all experiments except when cultured in collagen type 1‐coated plates (Falcon). In vitro calcification was induced by culturing VIC for 9 days in cell culture media supplemented with 2.6‐mM inorganic phosphate (Sigma), prepared as previously described (Carracedo, Artiach, et al., 2019 (link); Carracedo, Witasp, et al., 2019 (link)). For autophagy determination, VICs were stimulated for 2 h with bafilomycin A1 (100nM) and thoroughly washed with PBS before further treatments (Carracedo, Persson, et al., 2019 (link); Saliba‐Gustafsson et al., 2019 (link)). Viability was assessed after 24 h by WST‐1 reagent (Roche) according to manufacturer's protocol (Carracedo, Persson, et al., 2019 (link)).

In vitro calcification of VSMCs was induced by culturing cells in growth medium containing 3 mM inorganic phosphate (a mixture of NaH₂PO₄ and Na₂HPO₄, pH 7.4, Sigma) for up to 14 days, with a medium change every 3 days, as previously described [53] (link). The effects of glucocorticoids in FBS were assessed through comparison of charcoal-stripped and standard FBS (Life Technologies Ltd). Cells were treated with corticosterone (1–100 nM) (Sigma), 11-DHC (1–100 nM) (Steraloids, Newport, USA), carbenoxolone (10 μM) (Sigma), dexamethasone (1–100 nM) (Sigma), mifepristone (10 μM) (Sigma) or eplerenone (10 μM) (Sigma). The in vitro levels of corticosterone and 11-DHC used in the present study reflect those found in vivo. Plasma corticosterone levels in mice range from 20 nM (nadir, morning) to ~ 150 nM (peak, evening) and stress levels are typically 200–250 nM. Basal levels of plasma 11-DHC in mice have been reported at 2–5 nM and stress levels > 30 nM [12] (link).

VICs were seeded at 10,000 cells/cm² in 24-well plates (Corning New York, NY, USA). Calcification was induced by culturing VICs for 9 days in cell culture medium (DMEM, 5% fetal calf serum, 100 units/mL penicillin, 100 µg/mL streptomycin, 1 mM sodium pyruvate, 10 mM HEPES, and 2 mM L-glutamine) supplemented with 2.6 mM inorganic phosphate (Sigma, St. Louis, MO, USA).
Full-length recombinant human PRG4 (rhPRG4) was provided by Lubris BioPharma (Weston, MA, USA) [20 (link),21 (link)]. Dimethyl sulfoxide (DMSO) (vehicle) or rhPRG4 (10 µg/mL and 100 µg/mL) were added to the calcification medium and changed every other day. Calcification was assessed by IRDye^® 800CW BoneTag™ Optical Probe (Li-cor) according to manufacturer’s protocol. In brief, at 24 h before analysis, cells were incubated with the compound IRDye^® 800CW BoneTag™ Optical Probe (Li-cor) at a 1:10,000 concentration. After incubation, cells were washed with PBS and fixed with formaldehyde. Fluorescence was assessed and analyzed with an Odyssey CLx (Li-cor) near infrared imager.