Glycerophosphates

Adult flies were anesthetized by CO₂ and flash frozen in liquid N₂. Heads were separated from thoracicoabdominal segments, wings and legs by vigorous vortexing followed by separation over dry ice cooled sieves. In all, 600–10 000 frozen heads were added to 100 ml of 10 mM β-glycerophosphate pH 7, 2 mM MgCl₂, 5 mM sodium butyrate, 1X complete protease inhibitor cocktail (Roche: 11873580001), and the suspension was passed over a Yamato continuous flow homogenizer, set at 100 rpm, five to seven times. The homogenate was filtered over Miracloth (EMD Biosciences: 475855) and brought to 0.7 mM β-mercaptoethanol and 0.5% NP-40. After six tractions in two 40 ml Dounce homogenizers (tight-pestle B), 600 μl of antibody-adsorbed beads were added to 100 ml of lysate. The binding reaction was performed at 4°C for 30 min with constant end-over-end agitation. Beads were then collected on a magnet (Invitrogen: 123-02D) and washed three to four times in 50 ml 10 mM β-glycerophosphate pH 7, 250 mM sucrose, 2 mM MgCl₂, 25 mM KCl and 5 mM sodium butyrate. Bead-bound nuclei in 20 ml of wash buffer were then passed over a 20 um nylon mesh (Small Parts: B001D8ECDE), returned to the magnet stand and resuspended in 1 ml of 10 mM β-glycerophosphate pH 7, 250 mM sucrose, 2 mM MgCl₂, 25 mM KCl and 5 mM sodium butyrate. Sodium butyrate and the protease inhibitor cocktail are omitted from all buffers, if nuclei were to be used for transcript profiling (RNA-seq).

Human VICs were isolated from AV tissue, collected from patients undergoing AV replacement procedures (see Supplementary material online, Methods and Table 1). The study protocol was approved by the Institutional Review Board and Human Research Committee at Brigham and Women’s Hospital (2011P001703) including 10 donors. VICs were expanded in normal growth media (GM) composed of DMEM supplemented with 10% FBS and 1% P/S and then cultured in control media (CM), DMEM with 5% FBS and 1% P/S; or osteogenic media (OM), DMEM with 5% FBS, 1% P/S, 10 nM dexamethasone, 10 mM β-glycerol phosphate, and 100 mM L-ascorbate phosphate for 21 days (see Supplementary material online, Methods). Loss-of-function studies used small interfering RNA (siRNA) encapsulated in a lipid transfection agent to attenuate sortilin expression. VICs were processed for flow cytometry, quantitative polymerase chain reaction (qPCR), and western blot for VIC characterization at days 14 and 21 of culture (see Supplementary material online, Methods). VICs were lysed at day 14 to quantify ALP activity and Alizarin red density to quantify in vitro calcification. VICs were co-cultured with p38 MAPK inhibitor for 14 days and processed for qPCR. Liquid chromatography–tandem mass spectrometry for proteomics used the Thermo Fisher Oribitrap Fusion Lumos mass spectrometer with a Nanospray FLEX ion source and coupled to an Easy-nlC1000 HPLC pump (see Supplementary material online, Methods). Mass spectrometry data were analysed using Proteome Discoverer Package (Version 2.2, Thermo Fisher Scientific), and subsequent statistical analyses using Qlucore Omics Explorer. Pathway enrichment analysis was performed using ConsensusPathDB.^{20 (link)} Pathway networks were constructed using Python 3.8.1 and network visualizations were done in Gephi v0.9.2. scRNA-seq was conducted using the 10× Genomics platform. After cDNA library sequencing (Ilumina NovaSeq), raw data were analysed using Cell Ranger v3.1, Loupe v4.0, Seurat v4.0, R studio v.1.41717, and Excel.