Subsequently, monophosphate ions (PO43?) are transported into MVs through PiT1 located on the membrane of MVs, and the nucleation of calcium phosphate crystals is usually induced by PO43? and Ca2+ accumulated inside the MVs [47,48]. actin. In addition to ALP expression, the gene expression levels of type I collagen, osteopontin, elastin, fibrillin-1, and cluster of differentiation 90 are increased in ALP(+)-HUCPVCs. ALP(+)-HUCPVCs do not have the ability to induce mineralization nodules, which may be due to the restriction of phosphate uptake into matrix vesicles. Moreover, ALP(+)-HUCPVCs may produce anti-mineralization substances. We conclude that ALP(+)-HUCPVCs induced from HUCPVCs by a TGF-1 activation possess myofibroblast-like properties that have little mineralization-inducing ability. and that was generated on the basis of a mathematical model for relative quantification in the qPCR system. 2.5. Detection of Mineralized Nodules in HUCPVC HUCPVCs were grown on a 12-well plate at an initial density of 3.16 104 cells/cm2. After incubation for 24 h, Klf1 the medium was changed to a mineralization-inducing medium made up of 10 mM -glycerophosphate and 50 M ascorbic acid (mineralization medium) supplemented with or without TGF-1, VD, and LDN. The cells were cultured for up to 21 days. Mineralization was visualized by Alizarin Red S staining. After fixation with 4% paraformaldehyde neutral buffer answer for 10 min, the cells were stained with a 1% Alizarin Red S answer (Sigma-Aldrich) for 10 min, then washed with distilled water and photographed. This experiment was repeated 3 times, and the full image of all results that were cropped in the main physique is usually shown in Physique A2. 2.6. Characterization of Matrix Vesicles of HUCPVCs 2.6.1. Fractionation of Matrix Tioxolone Vesicles Matrix vesicles (MVs) were prepared by a modification to the method of Hayashi et al. [26]. HUCPVCs were grown on a 12-well plate at an initial density of 3.16 104 cells/cm2. After incubation for 24 h, the medium was changed to a mineralization-inducing medium and cultured with or without VD, LDN, and TGF-1 for 9 days. The cells were washed with 20 mM Tris-buffered saline (TBS) (pH 7.5) and digested with 0.45% collagenase for 1 h at 37 C. The digest was centrifuged at 2500 rpm for 10 min at 4 C and the pellet was designated whole cells or Cells. The supernatant was Tioxolone further centrifuged at 22, 000 rpm for 10 min at 4 C and the pellet was designated organelles or Org. The supernatant was finally centrifuged at 40, 000 rpm for 2 h at 4 C and the pellet was designated matrix vesicles or MVs. Three pellets, Cells, Org, and MVs were suspended with 20 mM TBS (100 L for Cells and 50 L for Org and MVs) and utilized for the experiments explained below. 2.6.2. ALP Activity Assay Three pellets (5 L each) in 96-well plates were mixed with 100 L of ALP reaction solution explained in Section 2.1. and incubated for 6 min at 37 C. We added 0.2 M NaOH (50 L) to quench the reaction, and the absorbance at 405 nm was then read on a plate reader. 2.6.3. Measurement of Ca Content Three pellets (5 L each) were mixed with 2.5 L of 0.5 N HCl in a tube and left to stand for 10 min. Samples were centrifuged at 13,000 rpm for 5 min at 4 C and the supernatant (2.5 L) was transferred into a 96-well plate. The calcium concentration in the supernatant was spectrophotometrically decided at 595 nm by following the color development with a calcium assay kit (Calcium E-test Wako, Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan). All values were normalized against the cultivation area. 2.6.4. Western Tioxolone Blotting SDS-PAGE was performed using 5C20% e-PAGEL mini gel (ATTO Corporation, Tokyo, Japan) electrotransferred onto PVDF membrane (Thermo Fisher Scientific). Following blocking with Blocking One answer (ATTO Corporation) for 1 h at room heat, polyclonal anti-PiT1/SLC20A1 antibody (GTX105062, Gene Tex, Irvine, CA, USA) was used at a dilution of 1 1:500 and reacted overnight at 4 C. The membrane was reacted against goat anti-rabbit IgG (BioRad, Hercules, CA, USA) at a dilution of 1 1:2000 for 1 h at room heat. The membrane was immunostained by chemiluminescent detection with ECL Prime (GE Healthcare. Uppsala, Sweden). The full image of the blots that were cropped in the main figure is shown in Physique A2. The band intensity was calculated using ImageJ software Version 1.52a (National Institutes of Health, Bethesda, MD, USA). 2.7. Search for Calcification Inhibitor Produced by HUCPVCs A porcine dental pulp-derived cell collection (PPU7) that we previously established [24] was used for this experiment. The FBS contained in the medium for this study was deactivated as explained in 2.1 Alkaline Phosphatase (ALP) Activity Assay. 2.7.1. Preparation of Conditioned Medium from PPU7 and HUCPVCs The PPU7 or HUCPVCs were separately plated on a culture.