5 fluorouracil

Biodiagnostic kits were purchased from Biodiagnostic Co. (Dokki, Giza, Egypt) for measurement of AST, ALT, urea, uric acid, creatinine, total cholesterol, HDL, LDL, MDA, leptin, insulin, glucose, α-amylase, BChe, and CAT levels. The enzyme α-glucosidase was purchased from Oriental Yeast Co. (Tokyo, Japan), while HEPES for making buffer solution was purchased from EMD Millipore Corp (Billerica, MA USA). Phenolic standards, i.e., luteolin, apigenin, phloretin, and phloridzin, and 5-fluorouracil as reference cytotoxic drug were purchased from Wako Pure Chemical Industries (Osaka, Japan). Orly as a reference anti-obese drug for in vivo experiment was obtained from Eva Pharma, Egypt. Acarbose as a reference antidiabetic for in vitro experiments was purchased from Wako Pure Chemical Industries (Tokyo, Japan).

Antibodies specific for TIE-1 (c-18) and TFIIH were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). KLF5 antibody was purchased from Abcam (Cambridge, MA, USA). Antibodies specific for XPC, cleaved-PARP, phospho-histone H2A.X (ser 139), and H2A.X were purchased from Cell Signaling Technology (Boston, MA, USA). Cc3-conjugated V5 antibody and β-actin antibody (A5441) were from Sigma (St. Louis, MO, USA). Hoechst 33342 was obtained from Dojindo (Kumamoto, Japan). Cisplatin, adriamycin, paclitaxel, 5-fluorouracil, methotrexate, carboplatin, and gemcitabine were purchased from Wako (Japan). Human TIE-1 cDNA was purchased from Kazusa DNA Research Institute (Kisarazu, Japan). Ovarian cancer cell lines were from the American Type Culture Collection. SiRNAs of TIE-1 (s14140, s14141), TIE-2 (s13983), XPC (s14929, s14930) and CSB (s 4806, s4807) are from Thermo fisher Scientific (Waltham, MA U.S.A.).

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).

Tenovin-6 was purchased from Cayman Chemical Company (Ann Arbor, MI). Docetaxel, SN-38, cisplatin, 5-fluorouracil (5-FU), doxorubicin and thapsigargin were obtained from Wako (Osaka, Japan). They were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mM and aliquots were stored at -20 °C. Stock solutions were diluted to the desired final concentrations with growth medium prior to use.

Human leukemia K562 and CCRF-CEM cell lines were grown in RPMI medium 1640 (1x, 11,875–093; Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) which included 10% fetal bovine serum (100–106; Gemini Bio-Products Inc., West Sacramento, CA, USA) and 1% penicillin-streptomycin (15140–122, Gibco) at 37 °C in a 5% CO₂ atmosphere. SLFN11-knockout cells were generated from CCRF-CEM and genetically modified K562 cell lines (K562 + vector and K562 + SLFN11) as described previously [18 (link), 20 (link)]. Nedaplatin (143–09481), carboplatin (033–25,231) and 5-fluorouracil (068–01401) were obtained from Fujifilm Wako Pure Chemical Corp. (Osaka, Japan).