Cell lineage and cell culture
Human lung epithelial cell line, BEAS-2B (CRL-9609), was obtained from ATCC (Manassas, VA, USA) and cultured using a Bronchial Epithelial Cell Growth Medium Bullet Kit (Catalog No. CC-3170, Lonza, Basel, Switzerland). Human Lung adenocarcinoma cell lines, A549 (CCL-185) was obtained from ATCC and cultured in F-12K Medium (Gibco, Waltham, MA, USA) added with 10% FBS (Invitrogen, Waltham, MA, USA). H1975 and HCC827 was obtained from ATCC (CRL-5908 and CRL-2868) and cultured in RPMI-1640 medium (Gibco) added with 10% FBS (Invitrogen). All the cells were cultured at 37°C in 5% CO2. For the PI3K/AKT/mTOR signaling activation, cells were incubated with the agonist 740Y-P (30 μM) for 24 h.
ID2 overexpression was achieved by transfecting ID2-overexpressing vector (ID2 OE) based on pLVX-puro plasmid. Short hairpin RNA cloned into pLVX-shRNA2 plasmid to against ID2 (sh-ID2) was transfected for ID2 knockdown. Blank vector (vector) and non-targeting sequence (sh-NC) were used as negative control, respectively. All transfections were performed with the help of Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA).
The cells and tissues were lyzed in RIPA buffer with ultrasonic probe. Use the bicinchoninic acid protein assay (BCA, Beyotime, China) was applied for determining the concentration of the protein samples. Protein samples (40 μg per lane) were separated using the Tris-glycine SDS-PAGE (8% to 15%) and then transferred onto PVDF membranes (Sigma-Aldrich, St. Louis, MO, USA). After blocking the non-specific bindings, the membranes were incubated with the following primary antibodies: ID2 (ab90055, Abcam, Cambridge, MA, USA), N-cadherin (ab76011, Abcam), E-cadherin (20874-1-AP, Proteintech, Wuhan, China), MMP2 (ab92536, Abcam), MMP9 (CSB-PA058909, Cusabio, Wuhan, China), AKT (Y409094, ABM, Vancouver, Canada), p-AKT (66444-1-1g, Proteintech), mTOR (ab2732, Abcam), p-mTOR (ab109268, Abcam) overnight at 4°C. Then, the primary antibody was detected with goat anti-mouse antibody conjugated with horseradish peroxidase. Enhanced Chemiluminescence Kit (ECL, Pierce) was used for signal visualization.
Cell Counting Kit 8 (CCK-8) assay
Use a CCK-8 kit (Sigma-Aldrich) to evaluate cell viability. Target cells were plated in the 96-well plates at a density of 5×103 cells/well and incubated for 24 h. After 48 h of transfection. 200 μl of CCK-8 solution was added to each well and incubated for another 3 h at 37°C. At the end of the incubation, cells were washed and the optical density (OD) was examined at 450 nm using a microplate reader (Thermo Fisher Scientific).
Cells, transfected or non-transfected, were cultured in a 6-well plate with standard culture medium overnight. Fourteen days later, all the cells were fixed with methanol and stained with crystal violet (0.1%). Finally, count the colonies under an optical microscope.
Transwell for cell migration
Transwell permeable support (8-μm, Corning, Tewkesbury, USA) was used for cell migration. Cells, transfected for 48 h or non-transfected, were suspended in serum-free medium into the upper chamber, culture medium containing 10% FBS was added to the lower chamber. After 24 h of incubation, the cells stayed on the upper surface of the membrane (non-invasive or non-migrating) were removed. Cells on the bottom surface of the membrane (migratory) were fixed with 4% paraformaldehyde for 10 min, and stained with 0.4% crystal violet solution. The migratory cells were imaged using a digital microscopy (Olympus, Tokyo, Japan).
A total of 15 paired Lung adenocarcinoma and adjacent non-cancerous tissues were obtained from patients who underwent lung cancer surgery in Xiangya Hospital, of Central South University. All the samples were immediately stored in a liquid nitrogen tank for long-term preservation. All patients included in this work or their relatives signed informed consent forms. This study was approved by the Ethics Committee of the Xiangya Hospital, of Central South University.
After section, tissue samples were placed in 10% paraformaldehyde and fixed overnight, processed, and embedded in paraffin. Tissues were then cut into 4-μm slices, dewaxed, and applied for Hematoxylin and eosin (H&E) staining or Immunohistochemical (IHC) staining. For IHC staining, sections were incubated with anti-ID2 (ab90055, Abcam) for 1 h at 37°C, then incubated with biotinylated goat anti-mouse IgG secondary antibody (Boster, China) for 20 min. SABC (streptavidin-biotin complex) was added to the samples and incubated at 37°C for 20 min. 3'-diaminobenzidine (DAB) color was permitted to develop for 5 min and samples were counterstained using hematoxylin. The slices visualized and quantified using an Olympus microscope.
At least three parallel experiments were performed for each experiment. All data were processed using the GraphPad software (San Diego, CA, USA) and shown as mean±standard deviation (S.D.). One-way analysis of variance (ANOVA) followed by a Tukey's multiple comparison test or Student's t-test were used to analyze the statistical significance of the differences. P value<0.05 is considered statistically significant.