2.1 Cell lines and reagents
Human lung cancer cells A549 and PC-9 were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in RPMI-1640 (Gibico, MA, USA) supplemented with 10% fetal bovine serum (Gibco, MA, USA). FAK inhibitor Y15 and AKT inhibitor 3CAI were purchased from MCM (NJ, USA). Pep2-Ae was purchased from Solarbio (Beijing, China). Cisplatin (Cis) and paclitaxel (PTX) were purchased from Sigma (NJ, USA).
2.2 Cell proliferation analysis
Cell proliferation was detected using the CCK8 kit (Biyuntian, Beijing, China). Briefly, 1 × 103 treated A549 or PC-9 cells were seeded into 96-well culture plates. 20 μl of CCK-8 solution was added into the 96 wells in determined time points. After 37 ℃ incubation of 2 hours, absorbance was measured at 450 nm on a microplate reader (Bio-Rad, MA, USA). Each experiment was performed for independent three times.
2.3 Colony formation
For colony formation assay, A549 or PC-9 cells (200 cells per well) were seeded into the 6-well plates and cultured at 37 °C for 14 days. After that, the colonies were fixed by 4% paraformaldehyde and stained by crystal violet. Colonies were pictured and counted. Each experiment was repeated independently in triplicate.
2.4 Transwell analysis
A549 or PC-9 cells (1 × 105 cells) were seeded in the upper transwell chamber (8 μm, Corning, CA, USA). The bottom chamber was filled with 0.5 ml medium containing 20% FBS. After 24 h, cells were fixed with 4% paraformaldehyde, and then stained with 0.05% crystal violet. The cells numbers were count. Each experiment was repeated independently in triplicate.
2.5 Western blotting
The protein lysates from A549 and PC-9 cells were separated by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membrane was incubated with the primary antibodies against to anti-p-FAK (1:1000, Abcam, Cambridge, UK), anti-t-FAK (1:1000, Abcam, Cambridge, UK), anti-p-AKT (1:1000, Abcam, Cambridge, UK), anti-t-AKT (1:1000, Abcam, Cambridge, UK), anti-FOXO1 (1:1000, Abcam, Cambridge, UK), anti-SOX2 (1:1000, Abcam, Cambridge, UK), anti-TRIB3 (1:1000, Abcam, Cambridge, UK) and anti-β-actin (1:1000, Abcam, Cambridge, UK), followed by incubation with an HRP-conjugated secondary antibody (1:1000, Abcam, Cambridge, UK).
2.6 Cytotoxicity analysis
The cytotoxicity of A549 or PC-9 cells to chemotherapy or inhibitor was analyzed using the FITC-Annexin V/ PE-PI apoptosis detection kit (BD, NJ, USA). Briefly, agents treated tumor cells were resuspended and stained with FITC-Annexin V and PE-PI staining solution for 15 min. Then cells apoptosis was detected by flow cytometry on a C6 flow cytometer (BD, NJ, USA). Each experiment was repeated for three independent times.
2.7 Animal protocols
Female NOD-SCID mice (6~8 weeks) were purchased from Huafukang (Beijing, China). All mice were housed in a specific pathogen-free facility. All animal experiments were performed according to the guidelines approved by the Institute Ethics Committee of Tianjin Medical University General Hospital. For subcutaneous lung cancer model, 106 A549 cells (50 μl PBS) were subcutaneously injected into NOD-SCID mice. After two weeks, mice were treated with PBS, PTX (5 mg/kg), Cis (5 mg/kg) and Pep2-Ae (10 mg/kg) by tail vein injection every two days. The tumor volumes were of mice were recorded every day (n=6). Survival was recorded on a daily basis (n = 6). The calculation formula of tumor volume: tumor volume = length × width 2/2. For tumorigenesis analysis, 105 A549 cells (50 μl PBS) or PC-3 cells (50 μl PBS) were subcutaneously injected into NOD-SCID mice. After 30 days, the tumor-bearing mice were counted. Each experiment was repeated independently in triplicate.
2.8 statistical analysis
The TCGA data were downloaded from http://ualcan.path.uab.edu/index.html and https://www.cbioportal.org/. Each experiment was performed for at least three independent times. Results were presented as the mean ± SEM and the statistical significance was analyzed using GraphPad 6.0 software (La Jolla, CA, USA). Statistical significance between groups was calculated by Student’s t test for two groups or by one-way ANOVA for more than two groups. The survival rates were determined by Kaplan–Meier survival analysis, *p < 0.05; **p < 0.01; ns, no significant difference.