Warfarin

Test compounds used in this study are listed in Table 1. These tested compounds are known to be teratogens inducing cleft palate in mammals and have been classified into various categories as a result of being tested in zebrafish experiments or chemical safety assays (Hillegass et al., 2008 (link); Selderslaghs et al., 2009 (link); Ito and Handa, 2012 (link); Lee et al., 2012 (link); Teixido et al., 2013 (link); Yamashita et al., 2014 (link); Inoue et al., 2016 (link); Martinez et al., 2018 (link); Cassar et al., 2019 (link)). The test compounds and exposure concentrations were determined based on Liu et al., 2020 (link). The exposure concentrations were as follows: hydroxyurea (1 mM, Sigma-Aldrich), valproic acid (7.5–30 μM, Wako), salicylic acid (100–400 μM, Wako), boric acid (1 mM, Wako), and caffeine (0.5–2 mM, Wako), which were diluted from stock solutions prepared with distilled water (Life Technologies), and imatinib (250 μM, Tokyo Chemical Industry), retinoic acid (10–50 nM, Tokyo Chemical Industry), thalidomide (400 μM, Tocris Bioscience), methotrexate (50–200 μM, Wako), warfarin (15–60 μM, Wako), phenytoin (1 mM, Wako), dexamethasone (1 mM, Wako), 5-fluorouracil (1 mM, Wako), and isoniazid (1 mM, LKT Laboratories), which were diluted from stock solutions prepared with dimethyl sulfoxide (DMSO, Wako).

Betamethasone, amlodipine, insulin, lansoprazole, loxoprofen, metformin and warfarin were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); allopurinol, famotidine, magnesium oxide and D-pantothenic acid were obtained from Nacalai Tesque, Inc. (Kyoto, Japan); aspirin was obtained from Merck Hoei Ltd. (Osaka, Japan); candesartan was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada); and rebamipide was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All other chemicals used were of analytical grade.

The MS-5 cells [25 (link)], OP-9 cells, distributed by RIKEN BRC through the National Bio-Resource of the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and MC3T3-E1 cells, were maintained as described [10 (link)]. Mouse BM-derived primary mesenchymal stem/stromal cells (MSCs; Gibco), and Med1^+/+ and Med1^−/− mouse embryonic fibroblasts (MEFs) that harbored a p53 inactivation [10 (link)], were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) [10 (link)]. The MB-1 niche-dependent myeloblastoma cells [26 (link), 27 (link)] were maintained by coculturing with mitomycin C (MMC)-treated OP-9 cells and used by coculturing with MMC-treated either MS-5 or OP-9 cells [12 (link)]. In some experiments, 0.5 μM LDN-193189 (Selleck) or 10 μM warfarin (Wako) was added to the coculture [24 (link)].