Bkm120

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].

The breast cancer cell lines MDA-MB-231 (triple negative), HCC1937 (triple negative), MCF-7 (ER-positive) and SK-BR3 (HER2-positive) were purchased from American Type Culture Collection (ATCC) (Manassas, VA). MDA-MB-231 and MCF-7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), HCC1937 cells were cultured in RPMI140 and SK-BR3 cells were cultured in MaCoy's 5A with 10% fetal bovine serum and antibiotics. The cells were incubated at 37°C under 5% CO₂. The compounds used in this study were purchased from the following vendors: Wortmannin (Sigma-Aldrich, St. Louis, MO), GDC-0941 (LC Laboratories, Woburn, MA), Perifosine (LC Laboratories, Woburn, MA), Everolimus (Sigma-Aldrich, St. Louis, MO), NVP-BEZ235 (LC Laboratories, Woburn, MA), BKM120 (Chemie Tek, Indianapolis, IN), XL147 (Chemie Tek, Indianapolis, IN), LGK974 (StemRd, Burlingame, CA), and WNT3A (R&D Systems, Minneapolis, MN).

The human CRC‐derived cell lines DLD1, HCT116 and LoVo were purchased from the American Type Culture Collection and were maintained in RPMI‐1640 culture medium (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; WelGENE) and 1% penicillin‐streptomycin solution (WelGENE) at 37°C in a humidified atmosphere with 5% CO₂. Cetuximab (C225; Erbitux; purchased from Merck, Darmstadt, Germany) was used at a final concentration of 5 mg/mL. BKM120 (200 mg) was purchased from Chemie Tek (Indianapolis, IN).