L ascorbic acid

TS^mole cells were established as described previously (14 (link)). Briefly, CT cells were isolated from CHM tissues and cultured on plates coated with 5–10 µg/mL Col IV (Corning) using TS medium (DMEM/F12 [Wako] supplemented with 0.1 mM 2-mercaptoethanol [Wako], 0.2% FBS [Thermo Fisher Scientific], 0.5% Penicillin-Streptomycin [Thermo Fisher Scientific], 0.3% BSA [Wako], 1% ITS-X supplement [Wako], 1.5 μg/mL l-ascorbic acid [Wako], 50 ng/mL EGF [Wako], 2 μM CHIR99021 [Wako], 0.5 μM A83-01 [Wako], 1 μM SB431542 [Wako], 0.8 mM VPA [Wako], and 5 μM Y27632 [Wako]). TS^bip #1, #2, and #3 were established in our previous study (14 (link)) and correspond to TS^CT #1, #2, and #3, respectively. TS^bip #4 was established in this study and used only for CNV analysis. Unless otherwise noted, we used TS^mole and TS^bip cells passaged 10–20 times for the analysis.

Fenquinotrione and its derivatives and metabolites were synthesized by the Kumiai Chemical Industry Co., Ltd. (Shizuoka, Japan). The structure of fenquinotrione, nuclear magnetic resonance (NMR) data, and mass spectrometry (MS) data for authentic standards are shown in Table 1. Three ¹⁴C-labeled compounds of fenquinotrione were used in the metabolic study: a 1-position label of a cyclohexenyl moiety (specific activity 4.94 MBq/mg, radiochemical purity 98.3%, abbreviated as [Cy-¹⁴C] FQ) synthesized by the Institute of Isotopes Co., Ltd. (Budapest, Hungary); the uniform label of a chlorophenyl ring (specific activity 5.63 MBq/mg, radiochemical purity 99.2%, abbreviated as [Qu-¹⁴C] FQ); and the uniform label of a phenyl ring (specific activity 5.29 MBq/mg, radiochemical purity 99.6%, abbreviated as [Bz-¹⁴C] FQ) synthesized by the Sekisui Medical Co., Ltd. (Ibaraki, Japan). The active form of benzobicyclon was synthesized by the Kumiai Chemical Industry Co., Ltd. Tefuryltrione, HPP, L(+)-ascorbic acid, iron(II) sulfate heptahydrate (FeSO₄·7H₂O), and isopropylthio-β-galactoside (IPTG) were purchased from the FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Rice plants (Oryza sativa L. var. Kinmaze) and Arabidopsis plants (Arabidopsis thaliana, ecotype Columbia-0) were used in this study.

Cultured cells were treated with osteoblast induction medium containing 10% FBS, 1% penicillin–streptomycin, 50-μg/mL L-ascorbic acid (Wako Chemicals GmbH ,Germany), 10-nM b-glycerophosphate (Sigma-Aldrich, Germany), 10-nM calcitriol (Sigma-Aldrich), and 10-nM dexamethasone (Sigma-Aldrich, Germany). Treatment was maintained for 14 and 21 days before proceeding with any analysis.

Stimulated secretion and decellularization were performed as previously reported [34 (link),36 ]. Briefly, the APS and RPS electrospun membranes were sliced and fixed on 2.2 cm × 2.2 cm slides, irradiated, and sterilized in six-well plates. Electrospun materials were coated with 0.2% gelatin (G7041, Sigma) for 4 h and rinsed thrice using PBS. The trypsin-digested hUMSCs (P2) at 90% confluence were grown on electrospun material surfaces at a density of 8000 cells/cm², and the hUMSCs medium (RP02010, Nuwacell) used to maintain cell proliferation. After the cells had reached 100% confluence, the medium was replaced with a hUMSCs medium supplemented with 50 μM l-ascorbic acid (014-04801, Wako) to stimulate ECM secretion for three days. To obtain cell-free material, the medium was discarded and the cells were rinsed using PBS. The cells were incubated in a decellularization buffer (0.5% Triton X-100 and 20 mM ammonia dissolved in PBS) and incubated at 37 °C for 5 min. After treatment with 100 U/mL DNAase I (D5025, Sigma) for 2 h at 37 °C, the cells were washed thrice using PBS to obtain MAPS and MRPS. The decellularized materials were aseptically stored at 4 °C for further analyses.

Single-stranded M13mp18 viral DNA, T4-βGT (10 units/μl), MspI (20 units/μL) and uridine-diphosphoglucose (UDP-Glc) were purchased from New England Biolabs (Ipswich, MA, USA). TET1 was purchased from Wisegene (Chicago, IL, USA). ARP [aldehyde reactive probe; O-(biotinylcarbazoylmethyl) hydroxylamine] was purchased from Cayman Chemical (Ann Arbor, MI, USA). Recombinant human TDG protein was purchased from Novus Biologicals (Centennial, CO, USA). Real-time PCR mix was purchased from Takara (Kyoto, Japan). All short oligonucleotides including staple DNAs were purchased from Eurofins Genomics (Tokyo Japan). Chemically modified DNA oligonucleotides were obtained from Japan Bioservices (Saitama, Japan). Ammonium iron (II) sulfate hexahydrate, l-ascorbic acid, α-ketoglutarate, dl-dithiothreitol, adenosine triphosphate, 25% glutaraldehyde acid were purchased from Wako pure chemical industries (Kyoto, Japan). 1 M HEPES buffer (pH 7.5), 1 M Tris buffer (pH 7.6) were obtained from Sigma-Aldrich. The buffer 10× NEB 4 [500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 10 mM DTT], and 10× cut smart buffer [500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 1 mg/ml BSA] were provided by New England Biolabs.