Gsk2334470

Embryonic DRG tissue from E13.5 mice was dissociated in trypsin–EDTA (Thermo Fisher Scientific, 25300054) and cultured on a dish as previously described [12 (link), 17 (link)]. The tissue culture plates were then coated with poly-d-lysine (Sigma, P0899) and laminin (Thermo Fisher Scientific, 23017015). The culture medium consisted of Neurobasal Medium (Thermo Fisher Scientific, 21103049) supplemented with 2% B-27 (Thermo Fisher Scientific, 17504044), 1% Glutamax (Thermo Fisher Scientific, 35050061), 1 μM 5-fluoro-2′-deoxyuridine (Sigma, F0503), 1 μM uridine (Sigma, U3003), 1% penicillin–streptomycin (Thermo Fisher Scientific, 15070063), and 50 ng/mL 2.5S nerve growth factor (Envigo, BT-5017). To knock down PDK1, the indicated lentivirus was added to the culture at DIV2. Cultured neurons were re-plated at DIV5 and then incubated at 37 °C in 5% CO₂ for 5 min with DMEM (Hyclone, 500mlsh30243.01)/0.05% trypsin–EDTA mixture (1:1). Cells were dissociated using gently pipetting, plated to new culture plates, and incubated for 14 h at 37 °C in 5% CO₂. The re-plated cells were fixed for 15 min in 4% paraformaldehyde and subjected to immunocytochemistry using anti-SCG10 and TUJ1 antibodies. Axonal lengths were measured using ImageJ. GSK2334470 (Selleckchem, S7087) was added in dissociated cells and mixed with pipetting.

Human nasopharyngeal carcinoma cells (CNE2, HONE1) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37°C. The approaches to modeled the resistant cell sublines was published before [53 (link)]. Acquired BEZ235 resistance were generated as described previously. Briefly, parental cells were continuously exposed to increasing doses of BEZ235 (from 0.05 μmol/L up to 0.4 μmol/L) for 6 to 9 months.
BEZ235, GSK2334470, JQ-1, decitabine and RG108 were purchased from Selleck Chemicals. Human normal IgG was obtained from Roche (Basel, Basel-Stadt, Switzerland). GAPDH antibody was obtained from Boster Biological Technology (Wuhan, China). MTT was purchased from Sigma-Aldrich (St. Louis, Missouri), and phospho-AKT, phospho-MYC, MYC, GSK3α/β antibodies were from Santa Cruz Biotechnology (Indian Gulch, California). All other antibodies were purchased from Cell Signaling Technology (Beverly, Massachusetts). Matrigel was purchased from BD Sciences. The Annexin V-FITC Apoptosis Detection Kit was from Invitrogen Inc. (Carlsbad, California).

Antibodies used in this study were anti-pAKT Ser473, anti-AKT, anti-pS6 Ser235/236, anti-S6, anti-pPRAS40 Thr246, anti-pERK1/2 Thr202/Tyr204, anti-ERK1/2, anti-p110α, anti-p110β, anti-PTEN, anti-IRS1 (Insulin receptor substrate), anti-poly (ADP-ribose) polymerase (anti-PARP), and anti-cleaved PARP from Cell Signaling Technology (Danvers, MA, USA), anti-phosphotyrosine 4G10 and anti-p85 from Millipore (Billerica MA, USA) anti-pIGF1R/IR Tyr1162/1163 from Biosource (ThermoFisher Scientific, Waltham MA, USA) and anti-IRS2 from Novus Biological (Littleton CO, USA). BYL719, AEW541, RAD001, and MEK162 were provided by Novartis Pharma AG (Basil, Switzerland). MK2206, AZD6482, GSK2334470, CAL101, and BMS354825 were purchased from Selleckchem (Houston TX, USA). All compounds used in vitro were dissolved in dimethyl sulfoxide (DMSO).

Human OS cell lines U2OS, SaOS2, HOS, 143b, and MG63 were purchased from ATCC (Manassas, VA). The LM‐7 OS cell line (Jia et al, 1999) was provided by Dr. Eugenie S. Kleinerman from the M.D. Anderson Cancer Center (University of Texas). 143b OS cells have been reported not to carry any EGFR mutations (Wen et al, 2007). 143b cells were cultured in DMEM supplemented with 10% FCS (Sigma), and LM7 cells were cultured in MEM with non‐essential amino acids, pyruvate, and 10% FCS (Sigma). Primary mouse OS cells were isolated and cultured as previously described (Grigoriadis et al, 1993). For EGFR inhibitor experiments, sub‐confluent cells were treated with indicated concentrations of erlotinib (Santa Cruz Biotechnology) for 24 h in normal growth medium (DMEM +10% FCS; Sigma). For in vitro stimulation experiments, cells were serum‐starved overnight and pre‐treated for 30 min with DMSO (1:1,000) or inhibitors against EGFR (5 μM afatinib, Santa Cruz Biotechnology), PDK‐1 (10 μM GSK2334470, Selleckchem), mTor (10 nM rapamycin, Sigma), or ERK1/2 (10 μM U0126, Cell Signaling Technology). Afterward, cells were stimulated for 0, 5, or 30 min with 50 ng/ml recombinant EGF (PeproTech). All cell lines were tested and showed no mycoplasma contamination.

TMD8 cells were obtained from the Tokyo Medical and Dental University, and OCI-LY10 cells were obtained from University Health Network. Both cell lines were cultured in RPMI-1640 medium supplemented with 20% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies, Carlsbad, CA). idelalisib and ibrutinib resistant TMD8 were cultured in the presence of idelalisib (1 μM) or ibrutinib (10–20 nM), respectively, and grown in a humidified atmosphere of 5% CO₂ and 95% air at 37°C. Compounds used in this study include: idelalisib (Gilead Sciences, Inc., Foster City, CA), GS-649443 (Gilead Sciences) [11 ], BYL719, AZD-6482, GDC-0941, MK-2206 and GSK2334470 (Selleckchem, Houston, TX) [12 (link)–16 (link)], ibrutinib (Shanghai Medicilon Inc., Shanghai, China), and ONO/GS-4059 (Ono Pharmaceutical Co., Trenton, NJ).