Methotrexate

The Screening Committee of Anticancer Drugs (SCADS) inhibitor kits I–IV (kit I v.3.2, kit II v.2.0, kit III v.1.5, kit IV v.2.3) were provided by the Ministry of Education, Culture, Sports, Science and Technology, Japan. Other inhibitors were obtained as follows: antimycin A (2247–10) (BioVision, Brugg, Switzerland), brequinar (B5707) (Tokyo Chemical Industry, Tokyo, Japan), atovaquone (AK544285) (Ark Pharm, Inc, Libertyville, IL, USA), teriflunomide (163,451–81-8) (Tokyo Chemical Industry), methotrexate (139–13,571) (FUJIFILM Wako Pure Chemical Corporation), cp466722 (25,417) (Cayman Chemical, MI, USA), etoposide (055–08431) (Wako), ve822 (24,198) (Cayman Chemical), rotenone (AK115691) (Ark Pharm, Inc), and NaN₃ (195–11,092) (FUJIFILM Wako Pure Chemical Corporation).

Dapsone, mitoxantrone, pitavastatin calcium, and lamotrigine were purchased from Toronto Research Chemicals (Toronto, Canada). Etoposide, methotrexate, carbamazepine, erlotinib hydrochloride, and verapamil hydrochloride were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Pefloxacin mesylate was purchased from LKT Laboratories (St. Paul, MN, USA). Nelfinavir methanesulfonate hydrate was purchased from Tokyo Chemical Industry (Tokyo, Japan). Moxalactam disodium salt was purchased from Sigma Aldrich (St. Louis, MO, USA). Dantrolene sodium salt was purchased from Cayman (Ann Arbor, MI, USA). Prazosin hydrochloride was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Quinidine was purchased from AOBIOUS (Gloucester, MA, USA). All marketed drugs not listed above and internal molecules defined as TK-1 to TK-43 were prepared by Takeda Pharmaceutical Company (Fujisawa, Japan). All other reagents and solvents were of analytical grade and were commercially available.

Tetrachloroauric (III) acid (HAuCl₄·3H₂O, 99.99%), methotrexate (MTX, 98%), dimethylformamide
(DMF, 99.5%), Lys (for biochemistry and sourced from egg white), methanol
(99.9%), RB, and heavy water (D₂O, 99.9%) were purchased
from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). We used
these chemicals without further purification. Pure water (resistivity:
18.2 MΩ·cm) was prepared using a Barnstead NANO pure DI
water system (Cole-Parmer Instrument Company, Vernon Hills, IL, USA).

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).

Infliximab (IFX), Etanercept (ETN), Certolizumab Pegol (CZP) were provided by Mitsubishi Tanabe Pharma Corporation (Osaka, Japan), Takeda Pharmaceutical Company Limited (Osaka, Japan) and UCB Japan (Tokyo, Japan), respectively. Methotrexate and folic acid were obtained from Wako Chemicals (Osaka, Japan). Rho kinase inhibitor, Y27632, and PKH-26 were supplied from Sigma (St. Louis, MO, United States). TO-PRO-3 was from Thermo Fisher Scientific (Waltham, MA, United States). Rabbit anti-phospho JNK monoclonal antibody (mAb) and anti-JNK polyclonal antibody (Ab) were purchased from cell signaling technology (Danvers, MA, United States). Anti-GAPDH Ab was from Gene Tex (Irvine, CA, United States).

HEK293T cells were cultured in Dulbecco's modified Eagle's Medium (D6546; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (S1820-500; Biowest/173012; Sigma-Aldrich), 50 U/ml penicillin, 50 μg/ml streptomycin (15070-063; GIBCO), and 2 mM glutamine (25030-081; GIBCO) in a 5% CO₂ incubator (hereafter referred to as normal culture conditions). For purine depletion experiments, cells were cultured in purine-depleted media, which is Dulbecco's modified Eagle's Medium supplemented with 10% dialyzed FBS (SH30079; Hyclone), 50 U/ml penicillin, 50 μg/ml streptomycin, and 2 mM glutamine. In the indicated experiments, cells were cultured with 2 μM methotrexate (135-13573; Wako), 10 μg/ml pepstatin A (4397-v; Peptide Institute), 10 μg/ml E64d (4321-v; Peptide Institute), hypoxanthine (086-03403; Wako), adenine (A0149; Tokyo Chemical Industry), guanine (G0169; Tokyo Chemical Industry), inosine (I4125; Sigma-Aldrich), or inosine 5'-monophosphate disodium salt hydrate (I0036; Tokyo Chemical Industry). Except for the screen, cells lacking de novo purine synthesis were maintained in media supplemented with 100 μM hypoxanthine, and the media were changed before analysis.