Thalidomide

Cisplatin (OncotecPharma production, Germany), pioglitazone hydrochloride, fenofibrate, thalidomide, GW9662, GW6471 (Tocris Bioscience, Bristol, UK) and thiopental sodium (Biochemie GmbH, Vienna, Austria) were purchased from commercial vendors.

Eight E3 ligase inhibitors namely, Thalidomide, ProTAME, NSC 66811, Nutlin 3, HLI 373, JNJ 26854165, SMER 3 and NSC 146109 were used in the study. Thalidomide targets cereblon (CRBN), which makes an E3 ubiquitin ligase complex with damaged DNA-binding protein 1 (DDB1) and cullin-4A (Cul4A). Thalidomide binds with CRBN and inhibits the associated ubiquitin ligase activity [6 (link), 7 (link)]. ProTAME, a cell-permeable prodrug, converts to an active molecule, tosyl-L-arginine methyl ester (TAME) and inhibits the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C) [8 (link), 9 (link)]. NSC 66811 and Nutlin 3 have been reported as MDM2 (Murine Double Minute 2) E3 ligase inhibitors [10 (link), 11 (link)]. JNJ 26854165 and HLI 373 are HDM2 (Human Double Minute 2) E3 ligase antagonists. SMER 3 is an inhibitor of a yeast SCF family E3 ubiquitin ligase (SCFMet30). Thalidomide, NSC6811, NSC 146109 and SMER 3 were purchased from Tocris Bioscience. Nutlin 3, JNJ 26854165 and ProTAME were purchased from Selleckchem, USA, Axon Medchem, Netherland and Boston Biochem, Inc. USA respectively. Chloroquine (CQ) was used as the positive control, and dimethyl sulfoxide (DMSO) was used as vehicle control in this study. CQ and DMSO were obtained from Sigma-Aldrich, 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).