Cell culture, cytokines, and inhibitors
Human embryo kidney 293 cells (HEK293) and the human lung adenocarcinoma cell line H2228, which harbors the EML4-ALK variant-3 fusion gene, were obtained from the American Type Culture Collection. The NIH3T3 mouse embryonic fibroblasts were obtained from the Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Gibco™ Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO2. All cytokines were purchased from PeproTech (USA), diluted as indicated by the manufacturer, and frozen in aliquots at -80°C. IL4 and IL6 were diluted in sterile ultrapure water. All inhibitors were purchased from Selleck Chemicals (USA) with dilution and storage according to the manufacturer.
Patients and samples
EML4-ALK-positive samples were obtained from seven NSCLC patients, as previously described [10] . The control group was patients with EML4-ALK-negative. Written informed consent was obtained from each patient and the work was approved by the Ethics Committee of Tianjin Medical University General Hospital. The demographicl and clinical characteristics of the EML4-ALK-positive patients used in study was showed in Table 1
Microarray gene expression analysis
Total RNA from H2228 cells was used to prepare biotinylated target cRNA using the Affymetrix One-cycle cDNA synthesis kit following the manufacturer’s instructions (Affymetrix, Santa Clara, CA).Then the biotinylated cRNA was fragmented and hybridized to the GeneChip Human Genome U133 plus 2.0 array(Affymetrix, Inc.), which contains more than 54,000 transcripts and expressed sequence tags.Raw Data were analyzed using Affymetrix GeneChip Operating Software (GCOS) 1.4 and filtered via 2-fold expression levels.P value cutoff of 0.05.Annotations were analysed using a combination of interactive query at NetAffx (www.affymetrix.com) and R suite.Clustering analysis made using MultiExperiment Viewer ( The Institute for Genomic Research, http://www.tigr.org/tdb/microarray/).
Immunohistochemistry and immunofluorescence
Immunohistochemistry and immunofluorescence were performed as previously described [10]. After being washed in PBS and blocked with 5% bovine serum albumin for 15 min at room temperature. Then sections were incubated the primary antibody against ALK (1:50, DAKO North America, USA), p-STAT3, p-STAT6, and p-JAK2(1:50,Cell Signaling Technology, Inc., USA) at 4°C overnight.Negative controls were set up by replacement of antibody with PBS.Cytoplasmic staining was considered positive for ALK.
Immunofluorescence labeling was performed as previously described[10]. Anti-ALK antibodies(DAKO North America, USA) were used at a dilution of 1:50, and p-JAK2, p-STAT3, and p-STAT6 antibodies(Cell Signaling Technology, Inc., USA) were used at a dilution of 1:100 for 2 h at 37°C. AlexaFluor 488-conjugated goat anti-rabbit (Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse were used as secondary antibody. Images were taken by an inverted fluorescence microscope (NIKON,Tokyo, Japan).
SiRNA and Plasmid construction
The EML4-ALK siRNA oligonucleotides (target sequence: CCTGTCAGCTCTTGAGTCA, positive-sense strand 5’- CCUGUCAGCUCUUGAGUCA- dTdT-3’, antisense strand 5’ -dTdT- GGACAGUCGAGAACUCAGU-3’) were synthesized at RiboBio (Guangzhou, China). A scrambled siRNA duplex was used as a negative control. The EML4-ALK variant-3 cDNA expression construct was engineered by cloning the EML4-ALK PCR product generated from H2228 into the pcDNA3.1 vector (Invitrogen) using the EcoRI and Xbal I sites (F:5'-CGGAATT CACTCTGTCGGTCCGCTGAATGAA-3'. R:5'-GCTCTAGACCAC GGTCTTAGGGATCCCAAGGAAGAGAA-3').
Protein extraction, immunoprecipitation, and western blotting
For the preparation of total cell lysates, cells were washed with PBS and lysed on ice for 30 min in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100) containing a protease inhibitor cocktail (Roche, Mannheim, Germany), 50 mM NaF, and 1 mM Na3VO4. The samples were cleared at 12000 rpm for 15 min at 4°C, and the proteins quantified using the BCA protein assay (Thermo Scientific, MA, USA) using bovine serum albumin (BSA) as a standard. Equal amounts of proteins (10 to 40 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes (Amersham Biosciences, NJ). After washing, membranes were incubated with primary antibody overnight at 4℃ under gentle rocking. Primary antibodies included anti-ALK,JAK2, STAT1, STAT3, STAT5, and STAT6 ,and anti-phospho-ALK,JAK2, STAT1, STAT3, STAT5, and STAT6 antibodies(all 1:1000 dilution), which were purchased from Cell Signaling Technology (Danvers, MA, USA).And then exposed to HRP-conjugated secondary antibody (1:1000 dilution, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. Bands were visualized using Pierce ECL Substrate (Thermo Fisher Scientific, Waltham, MA, USA).
Immunoprecipitation was performed on 800 μg of total protein by adding anti-JAK2 antibodies overnight at 4°C. The immune complexes were precipitated with Protein A-Sepharose 4B (Thermo Fisher Scientific, Waltham, MA, USA) for 2 h at 4°C. Immunoprecipitated proteins were washed, recovered by boiling in 5×SDS sample loading buffer, and then separated by SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes at 100 V for 60 min in transfer buffer (20 mM Tris, 150 mM glycine, 20% methanol) and probed with primary antibodies against ALK, JAK2 and phospho-STAT6.The following steps were done as above.
CCK-8 assay
The Cell Counting Kit-8 (Beyotime, Shanghai, China) was used according to the manufacturer’s instructions. Approximately,3×103 NIH3T3 or HEK293 cells in the exponential growth phase were seeded into 96-well plates and transiently transfected with pcDNA3.1-EML4-ALK-V3, EML4-ALK siRNA, control vector, or control siRNA for 24, 48, or 72 h. H2228 cells (1×104 cells/well) were cultured in 96-well plates for 24 h and then treated with TAE (0. 0.001, 0.01, 0.1, 1, 10, 100 μM) alone or in combination with SH-4-54 (0, 8, 12, 18, 27, 40, 0 μM) or ruxolitinib (0, 4, 16, 64, 256, 1024, 4096 μM) for an additional 24 h before performing the CCK8 assay.Then,10 μL of CCK8 solution was added into each well and incubated for 1 h. The absorbance of each well was quantified at 450 nm using a microplate reader (SpectraMax M5, Molecular Devices, CA, USA). All data were calculated from triplicate wells.
5-Ethynyl-2’-deoxyuridine (EdU) staining
Cells were stained with the Cell-Light™ EdU stain kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. Briefly, cells were cultured with 50 μM EdU for 2 h followed by two washes with PBS and fixation with 4% paraformaldehyde. After permeabilization with 0.5% Triton X-100 and washing with PBS, the cells were dyed with Apollo (Red) and Hoechst 33342 (Blue) in dark for 30 min and analyzed by fluorescence microscopy.
Flow cytometry analysis of apoptotic cells
H2228 cells (2×105cells/well) were seeded into 6-well plates and cultured for 24 h. The cells were transfected with siRNA-NC or siRNA-EML4-ALK and cultured for another 24 h. The transfected cells were stained using the Annexin V-FITC Apoptosis Analysis Kit (BD Biosciences, CA, USA) and analyzed by flow cytometry using a FACSAria™ flow cytometer (BD Biosciences, CA, USA).
Statistical analysis
All data were analyzed using the Statistical Package for Social Sciences (version 16.0., SPSS Inc., Chicago, Illinois, USA). The Student’s t-test was used to identify statistical significance between two experimental groups. Multivariate analysis of variance (MANOVA) was used to identify statistical significance for H2228 cells viability for the three inhibitor groups. P < 0.05 indicated statistical significance.