Tissue culture reagents

Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, and tissue culture reagents were obtained from Invitrogen/GIBCO (Grand Island, NY, USA). Momordicine I (>99% purity, kindly provided by Dr. Shi-Yie Cheng, Department of Life Sciences, College of Science, National University of Kaohsiung, Kaohsiung, Taiwan, ROC) was dissolved in dimethyl sulfoxide (DMSO), and the DMSO content in all groups was 0.1%. Anti-p-Smad2/3, anti-Smad2/3, anti-GAPDH, and anti-PARP antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-HO-1 and anti-Nrf2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Brusatol, the Nrf2-specific inhibitor, and all other reagent-grade chemicals were acquired from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Protein G–Sepharose was from GE Healthcare. [γ-³²P]ATP was from PerkinElmer. Agarose-conjugated anti-FLAG M2 antibody, Triton X-100, EDTA, EGTA, sodium orthovanadate, sodium glycerophosphate, sodium fluoride, sodium pyrophosphate, 2-mercaptoethanol, sucrose, benzamidine, Tween 20, Tris/HCl, sodium chloride, magnesium acetate and doxycyclin were from Sigma. PMSF was from Melford. Tissue culture reagents, Novex 4–12% Bis-Tris gels and NuPAGE LDS sample buffer was from Invitrogen. Ampicillin was from Merck. P81 phosphocellulose paper was from Whatman. Methanol and chloroform were from VWR Chemicals. Inhibitors GDC-0941 (Axon Medchem), GSK2334470 (Tocris), AZD8055 (Selleck) and BKM120 (Chemie Tek) were purchased from the indicated suppliers. VPS34-IN1 (1-[{2-[(2-chloropyridin-4yl)amino]-4′-(cyclopropylmethyl)-[4,5′-bipyrimidin]-2′-yl}amino]-2-methyl-propan-2-ol) was synthesized as described in patent WO 2012085815 A1 [Cornella Taracido, I., Harrington, E.M., Honda, A. and Keaney, E. (2012) Preparation of bipyrimidinamine derivatives for use as Vps34 inhibitors; method for synthesis of this compound is described on page 73, Table 4, example 16a.VPS34-IN1 has a CAS registry number 1383716-33-3].

In the presence of feeder layer, the HUES cells were cultured on feeder layer in knockout DMEM supplemented with 10% knockout serum replacement (KSR), 10% plasmanate, 1% PenStrep, 1% Glutamine, 1% nonessential amino acids, 10 ng/mL bFGF, and 55 μmol/L β-mercaptoethanol as described (Cowan et al., 2004 (link)). In the absence of feeder layer, HUES cells were cultured on matrigel-coated plates in the mTeSR1 medium with 5× supplement. All tissue culture reagents were purchased from Invitrogen.

Chemical reagents were purchased from Sigma-Aldrich (Oakville, Canada) and tissue culture reagents from Invitrogen (Burlington, Canada), unless stated otherwise. Dr. Roger Leng, University of Alberta, provided the p53 and PG null non-small cell lung carcinoma cell line H1299 [91 (link)]. The p53 mutant and PG deficient human tongue squamous cell carcinoma cell line SCC9 has been described [23 (link), 24 (link)]. All cells were maintained in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin-kanamycin (PSK) antibiotics.

Lymphoprep was purchased from Nycomed Pharma (Zürich, Switzerland) and tissue culture reagents from Gibco Invitrogen (Gaithersburg, MD, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).