BATF expression analysis in TCGA database
TCGA is a publicly funded project and contains RNA expression data for various types of cancer, including NSCLC (http://cancergenome.nih.gov/). The mRNA expression profiling data of NSCLC and matched adjacent mucosa was downloaded from TCGA (datasets TCGA-38, TCGA-49 and TCGA-50) (19).
Cell lines and culture conditions
The human NSCLC cell line A549 was purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin (Sigma-Aldrich; Merck KGaA) and 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA), at 37°C in a humidified incubator containing 5% CO2.
Reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was extracted from tissue samples and cultured cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. cDNA was synthesized from total RNA (500 ng) in a 10 µl reaction volume using a PrimeScript RT-PCR kit (Takara Bio, Inc.) using the following conditions: 5 min at 25°C, 25 min at 52°C and 10 min at 80°C. Then, qPCR was performed using the power SYBR Green master mix (Thermo Fisher Scientific, Inc.). GAPDH was used as an internal control. The primers used were: BATF forward, 5′-AGAAGAGTTCAGAGGAGGGA-3′ and reverse, 5′-CGTTCTGTTTCTCCAGGTCT-3′; GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′. qPCR was performed using SYBR Premix Ex Taq II (Takara Biotechnology Co., Ltd.), and the thermocycling conditions used were: 95°C for 10 min, followed by 39 cycles of 95°C for 1 min, 55°C for 30 sec, and 72°C for 45 sec. The 2−ΔΔCq method was used to determine relative (BATF/GAPDH) mRNA expression (20).
Lentiviral shRNA vector construction and infection
For knockdown of BATF, a lentiviral shRNA vector targeting the human BATF gene was constructed by Shanghai GeneChem Co., Ltd. The targeting sequence of BATF was synthesized and cloned into the lentiviral vector pGVX115-GFP (Shanghai GeneChem Co., Ltd.) to produce pGV115-shBATF at 37°C for 2 h. For control cells, transfection was performed using an empty vector carrying GFP. To establish stable cell lines, 2 µg/ml puromycin was added to the medium for 1 week following transfection with the lentiviral vectors. Knockdown efficiency was verified using RT-qPCR and western blot analysis.
Western blot analysis
Cell lines were harvested 3 days post transfection and washed twice with cold PBS, and then cell lysates were prepared by adding radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) containing 1 mM phenylmethanesulfonyl. The lysate was stored at −20°C until further experimentation. Proteins (~30 µg each lane) were separated with 12% SDS-PAGE, and transferred onto PVDF membranes (EMD Millipore; Merck KGaA). Membranes were incubated with primary antibodies to BATF (1:500; cat. no. ab236876; Abcam), and β-actin (1:500; cat. no. sc-47778; Santa Cruz Biotechnology) overnight at 4°C. Next, the membrane was washed and incubated with the corresponding secondary antibodies [horseradish peroxidase-conjugated (HRP) goat anti-rabbit; 1:5,000; cat. no. P044801-2; Dako; Agilent Technologies or HRP goat anti-mouse IgG H&L; 1:1,000; cat. no. ab6708; Abcam or goat anti-rabbit IgG; 1:1,500; cat. no. bs-0295G; BIOSS] at room temperature for 1 h. Protein bands were quantified using a bio-imaging system (DNR Bio-Imaging Systems, Ltd.).
Cell proliferation and viability assays
Cell proliferation and viability were analyzed by the Celigo imaging cytometry system and MTT assay, respectively. The fluorescence intensity of entire wells, of transfected A549 cells was detected, and the number of cells was automatically calculated by the Celigo imaging cytometry system (Nexcelom Bioscience LLC). Cell viability was measured using a Cell Viability kit (MTT; Roche Diagnostics) solubilized in 150 µl dimethyl sulfoxide according to the manufacturer's instructions. The absorption of the solution was measured at 570 nm at various time points.
Apoptosis assay
A total of 1×105 cells/well were incubated in 6-well plates for 24 h and treated with shBATF and shCtrl for 48 h, both at 37°C. The cells were washed, and the Annexin V-APC Apoptosis Detection kit (eBioscience, Inc.) was used according to the manufacturer's instructions. Cells were analyzed by flow cytometry (FACScalibur; BD Biosciences), and the percentage of apoptotic cells was determined using ModFit LT v5.0 software (Verity Software House, Inc.). Each experiment was performed three times.
Caspase 3/7 activity was measured using a homogeneous luminescence-based assay with a Caspase 3/7 Glo Assay kit (Promega Corporation) according to the manufacturer's instructions. Following transfection with shCtrl and shBATF, A549 cells were treated with 0, 0.01, 0.1 and 1.0 µM cetuximab for 24 h. For luminosity, the cells were analyzed by an Infinite F500 multifunction microplate reader (Tecan Trading AG).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software, Inc.). Statistical differences were analyzed and calculated by unpaired two-tailed Student's t-test or one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.