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AG1478 and CL387,785 were obtained from the Calbiochem Corporation (San Diego, CA, USA). EGF was purchased from Sigma-RBI (Natick, MA, USA). For the baculoviral expression vector of glutathione S-transferase (GST) tagged EGFR kinase domain (KD) with L858R/T790M double mutation (DM), pBac-PAK8-GST-EGFR(L858R/T790M)-KD, PCR amplified cDNA COX Inhibitors fragment containing human EGFR kinase domain from amino acids 696 to 1022 was attached to the C-terminal coding region (3? region) of the glutathione S-transferase gene and the fused DNA fragment is cloned into a baculovirus expression vector pBacPAK8 (Clontech, Palo Alto, CA, USA). The NSCLC cells lines H1975 and myeloid cell line 32D were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).
The H1975 cell line were maintained at 37?C under 5% CO2 in RPMI-1640 medium supplemented with 10% fetal calf serum plus penicillin (50 U/ml) and streptomycin (50 mg/ml). The myeloid cell line 32D and 32D-EGFR(L858R/T790M) were cultured at 37?C under 5% CO2 in RPMI-1640 medium supplemented Rapamycin with 10% fetal calf serum, IL-3 (3 ng/ml), L-glutamine (2 mM) plus penicillin (50 U/ml) and streptomycin (50 mg/ml). EGFR-expressing 32D cells, 32D-EGFR(L858R/T790M), were established as described previously (6, 7). High-throughput screening. For the 32D-EGFR(L858R/T790M) cell-based high-throughput screening, the cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, Madison, WI, USA).
The growth of 32D-EGFR(L858R/T790M) cells is expected to be EGF independent due to a mutation in the EGFR kinase domain that renders the kinase constitutively active (6). The addition of EGF was found to enhance cell survival compared with the IL3-dependent growth of cells. Given this Sunitinib observation, we also added EGF (at a concentration of 10 ng/ml) to the culture medium used for compound screening. In brief, 5,000 32D-EGFR(L858R/T790) cells in 100 ?l culture medium (containing 10 ng/ml EGF or 3 ng/ml IL-3) were seeded in each well of 96-well microtiter plate. At the end of the 72-h incubation with 10 ?M of each test chemical, cells in each well were incubated with 20 ?l of an MTS and phenazine methosulfate (PMS) mixture (MTS/PMS ratio: 20:1) for 2 h at 37?C in a humidified incubator with 5% CO2 to allow viable cells to convert the tetrazolium salt into formazan.
The amount of formazan produced was determined by measuring the absorbance at 490 nm using a PerkinElmer Victor2 plate reader (PerkinElmer, Shelton, CT, USA). Initial hits were identified that satisfied the following criteria, cell grown inhibition compared to vehicle control by compound was >70% under EGF stimulation, and <30% under IL-3 stimulation. Purified kinase confirmatory assays. The GST-EGFR(L858R/T790M)- KD recombinant proteins were expressed in Sf9 insect cells transfected with pBac-PAK8-GST-EGFR-KD plasmid. The Nilotinib purification procedure used for the GST-EGFR(L858RT/790M)-KD proteins was similar to that used for EGFR(WT)-KD proteins in our previous studies (4, 5).
The EGFR(DM) Kinase-Glo assays were carried out in 96-well plates at 37?C for 60 min in a final volume of 50 ?l including the following components: 200 ng GST-EGFR(L858R/T790M)-KD proteins, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 4 mM MnCl2, 2 mM dithiothreitol (DTT), 10 mM MgCl2, 0.1 mg/ml bovine serum albumin, 10 ?M poly(Glu, Tyr) 4:1, 0.5 mM Na3VO4, and 1 ?M ATP. Following incubation, 50 ?l Kinase-Glo Plus Reagent (Promega) was added, and the mixture was incubated at 25?C for 20 min. A 70-?l aliquot of each reaction mixture was transferred to a black microtiter plate, and the luminescence was measured using a Wallac Victor 1420 multilabel counter (PerkinElmer). The EGFR(WT) kinase assay was described in our previous paper (4). Western blot analysis.
For the H1975 immunoblot analysis, cells were plated and incubated for 16 h in complete RPMI-1640, then incubated with serum-free medium for another 24 h, and incubated with test compounds in serum-free medium for 2 h followed by stimulation with EGFR for 10 min. H1975 cultured cells were suspended by lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% dodecyl sulfate, 1 mM sodium orthovanadate, 1 mM phenylmethanesulfonylfluoride or phenylmethylsulfonylfluoride (PMSF), and 1 mM DTT). The cell lysates were kept on ice and incubated for 10 min. The lysate was cleared by centrifugation at 14,000 g for 15 min at 4?C. After adding 5? sample buffer, the supernatants were heated at 95?C for 5 min and cell extract samples (10 or 20 g) were Gemcitabine separated by 10% or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA).
After transfer to membrane, probing was carried out using the indicated primary antibodies from Cell Signaling Technology (Beverly, MA, USA) at a 1:1000 dilution: anti-EGFR, anti-pEGFR(Tyr845), antipEGFR( Tyr1068), anti-STAT3 anti-pSTAT3 (Tyr705), anti-STAT5b, anti-pSTAT5b (Tyr694), anti-Src, anti-pSrc(Tyr416). Anti-?-Actin primary antibody (Santa Cruz, CA, USA) was used at 1:10,000 dilution. The membrane was then developed using SuperSignal reagent (Pierce, Rockford, IL, USA) and exposed to X-ray film. Data analysis. The IC50 value was defined as the amount of compound that induced a 50% reduction in cell viability in comparison with dimethyl sulfoxide (DMSO) treatment controls and was calculated using GraphPad Prism version 4 software (San Diego, CA, USA). The Z? values were estimated from the sample means and sample standard deviations and calculated as defined by Zhang et al.; positive standard deviation, negative mean, and negative standard deviation, respectively.

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