Boric acid

The human breast adenocarcinoma (MCF-7) cell line was obtained from the Egyptian Holding Company for Biological Products and Vaccines (VACSERA, Giza, Egypt). Ammonium nitrate, magnesium sulfate, potassium nitrate, boric acid, cobalt chloride, cupric sulfate, manganese sulfate, potassium iodine, sodium molybdate, zinc sulfate, citric acid and sodium citrate were purchased from Wako Pure Chemical Industries^® (Tokyo, Japan). Calcium chloride, dextrose, potassium hexacyanoferrate II, potassium hexacyanoferrate III and sodium carbonate were obtained from Merck^® (Darmstadt, Germany), while potassium phosphate, sodium chloride, ferrous sulfate, Na₂-EDTA2H₂O, nicotinic acid, pyridoxineHCl, thiamineHCl, Fe-EDTA, 4-morpholineethanesulfonic acid, τ-inositol, sucrose, phytoagar, sodium phosphate, silymarin, triton X-100, 5-bromo-4-chloro-3-indoly-β-d-glucuronide and α-Rhamnase were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). 17β-estradiol was purchased from Takeda Chemical Industries Ltd. (Osaka, Japan). Solvents used were of analytical grade unless mentioned and were purchased from Merck, J.T. Backer^® (Deventer, The Netherlands) and Theo Seulberger^® (Karlsruhe, Germany). Media and supplements for cell cultures were obtained from Gibco^®/Invitrogen (Karlsruhe, Germany) and Greiner Labortechnik^® (Frickenhausen, Germany).

FL, lignoceric acid, palmitic acid, boric acid, potassium chloride, sodium hydroxide, chloroform, methanol, and ethanol were purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Cholesterol, cholesteryl sulfate, octacosanoic acid, 1,6-diphenyl-1,3,5-hexatriene (DPH), N,N,N-trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl)phenyl-ammonium p-toluenesulfonate (TMA-DPH), and 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS) were from Sigma-Aldrich (St. Louis, MO, USA). Ceramide type III and ceramide type VI were from Evonik Industries AG (Essen, Germany). These reagents were used without further purification.

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).

Thrombin from human plasma and apo-transferrin were purchased from Sigma-Aldrich (USA). BSA and fibrinogen from human plasma were purchased from Wako Pure Chemicals Industries (Japan). All ssDNAs, except for the random ssDNA library with its primer set, were synthesized by Sigma-Aldrich (USA). 2-Morpholinoethanesulfonic acid (MES; Wako, Japan), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimido hydrochloride (EDC; Wako, Japan), Dulbecco’s PBS(−) (Wako, Japan), 10% Tween 20 solution (Bio-Rad, USA), tris(hydroxymethyl)aminomethane (Tris; Wako, Japan), 1 M sodium chloride solution (Wako, Japan), 1 M magnesium chloride solution (Wako, Japan), 1 M sodium hydroxide solution (Agilent Technologies, USA), 0.5 M borate buffer at pH 8.5 ± 0.2 (Polysciences, USA), EDTA disodium salt dihydrate (Wako, Japan), and boric acid (Wako, Japan) were used as received. All solutions were prepared using ultrapure water from a Milli-Q water purification system (Merck Millipore, USA). For the preparation of gels, a 37.5:1 (40%, w/v) acrylamide/bis solution, 2.6% C (Serva Electrophoresis, Germany), ammonium persulphate (Bio-Rad, USA), and N,N,N’,N’-tetramethylethylene (TEMED; Bio-Rad, USA) were used. The loading buffer and a 25-bp DNA stepladder were purchased from Wako (Japan).