The PC9 and HCC4006 cells were collected from the American Type Culture Collection (Manassas, VA, USA). Lung cancer cells were cultured and acquired gefitinib resistance according to procedures described in our previous study (11).
Lentivirus construction and infection
The TRAF1 short hairpin(sh) RNA was purchased from the RNAi Consortium (Broad Institute, Cambridge, MA, USA). We purchased negative control and shTRAF1 plasmids (GV112) from GeneChem (Shanghai, China). Lentivirus construction and infection were carried out according the procedures reported in our previous study (11).
Cell proliferation assay
The cell proliferation assay was performed by counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). For the cell proliferation assay, 1000 cells/well were seeded in 96-well plates and cultured 24h. According to the manufacturer's instructions, cells were incubated with 10ul CCK8 reagent for 1h at 37°C, then assayed at 450nm on a Multiskan microplate reader.
Detection of apoptotic cells by flow cytometry
The annexin V-FITC/PI staining kit was purchased from Thermo Fisher Scientific. A FACS Aria II flow cytometer (BD Biosciences) was used to detected cells. The procedures were performed according the manufacturer's instructions and our previous study (12).
Cell cycle analysis
For the cell cycle analysis, 5 × 105 PC9R cells per well were seeded in the 6-well plates. We incubated the PC9R cells with gefitinib. According the manufacturer's instructions, the cells were harvested, fixed and stained. The flow cytometry was used to analysis.
The TRIzol reagent was obtained from Thermo Fisher Scientific. RT-PCR kit was purchased from Toyobo. The experimental procedures and results analysis were carried out according to the manufacturer's instructions.
PCR primers: TRAF1, 5'-CTACTGTTTTCCTTTACTTACTACACCTCAGA-3'
and 5'- ATCCAGACAACTGTTCAAACTGATG-3';
The anti-TRAF1 polyclonal antibody (sc1830) and β-actin antibody (A3854) were obtained from Santa Cruz Biotechnology and Sigma-Aldrich. The procedures of western blot (WB) were carried out according manufacturer's instructions and our previous studies (8).
Pleural effusion sample collection
The 50 patiens with EGFR TKIs sensitive and 45 patients with EGFR TKIs resistance were enrolled in the study at the Department of Pulmonary Medicine and Department of Oncology, Shengli Oilfield Central Hospital, Shandong Province, China. The study protocol and informed consent providing to the participants were verified by the institutional ethics committee. Clinic sample collection and data management were performed in conformity with guidelines. After collected the samples, in 1000 × G centrifugation for 10 min to obtain cell pellets. TRAF1 mRNA level was quantitated by qPCR.
All experiments were carried out with triplicate samples. SPSS version 20 and GraphPad Prism version 5.0 were used for statistical computation. The principles of statistical analysis were consistent with our previous studies. P < 0.05 means statistically significant.