Cell culture
The lung cancer cell line Calu-6 was bought from Chinese Academy of Sciences. Cells were preserved at 37°C and 5% CO2, maintained and expanded in growth medium containing high glucose DMEM (Gibco, USA) combined with 15% heated-inactivated fetal bovine serum (FBS, Zhengbo, Beijing, China) and 100 U/ml streptomycin/penicillin (Gibco, USA) for one to two weeks.
Construction of pLVX-AcGFP1-N1-S100A2
PLVX-AcGFP1-N1-S100A2 was generated by PCR method using pcDNA-S100A2. The primers were designed as follows: the forward primer was 5’-CCCAAGCTTACCATGTGCAGTTCTCTGGAGCAG-3’ and the reverse primer was 5’-GACGGGTCTGGCTGGGCCTTAAGGC-3’. DNA sequencing was then applied to verify the Orientation and the insert sequence.
Virus production and concentration
Plvx-acgfp1-n1-S100A2, pspax2 and pmd2. G were co-transfected into 293T cells with a ratio of 2:1:1. The fluorescence expression was observed under the microscope after 72 hours and the supernatant was collected prior to be filtered through a 0.45 µm PVDF filter (Millipore). Thirty-fold concentrated stock was obtained by ultracentrifugation (50 000 g, 150 min, 4°C). The pellets were resuspended in PBS and stored at -80°C.
Cells infection
Those Calu-6 cells grown in logarithmic phase were collected, trypsinized to a density of 3*105/ml and planted in 6-well plate (JET BIOFIL, Guangzhou, China), then cultured in incubator. S100A2 over-expressed lentivirus (MOI = 10) was added when cell confluence reached 80–90%. Meanwhile, we transfected Calu-6 cells with blank vector control lentivirus (MOI = 10) as one control group. It is also notable that another control group was empty Calu-6 cells with no vector. We designated all these three groups as Calu-6/S100A2, Calu-6/neo and Calu-6. The infection efficiency was observed by fluorescence microscopy after 48 hours, and the infected S100A2 gene was confirmed by subsequent experiments including QPCR and WB.
RNA extraction and QPCR
We applied TRIzol reagent (Invitrogen, USA) for cell lysis, then collected the centrifugal supernatant. To get purified RNA, chloroform, 2-propanol and 75% ethanol were successively added into the supernatant to separate RNA from DNA and protein. After the extraction, we used Nanodrop 2000 (Thermo, USA) to detect the concentration and purity of RNA. RevertAid First Strand cDNA Synthesis Kit (Thermo, USA) helped us achieve the process of reverse transcription, and cDNA was synthesized from 2 ml RNA by RevertAid™ M-MuLV, 10 mM dNTPs, 1 µl oligo (dT) 18 primers, reverse transcriptase, and 1 µl Ribolock™ ribonuclease inhibitor. All operations were strictly operated according to the kit instructions. The specific procedures and conditions in PCR were incubation (95°C, 5 min), denaturation (94°C, 30 s for 44 circles), annealing (55°C, 30 s), extension (72°C, 30 s). In addition to that, the information about primers used in PCR was as follows: S1002 forward primer (5-GCCAAGAGGGCGACAAGTT-3) and S1002 reversed primer (5-AGGAAAACAGCATACTCCTGGA-3); glyceraldehyde-3-phosphate dehydrogenase (GAPDH, an endogenous control) forward primer (5-GGAGCGAGATCCCTCCAAAAT-3) and GAPDH reversed primer (5-GGCTGTTGTCATACTTCTCATGG-3).
Western Blot
The western bolt experiment was used to determine the protein expression of Calu-6/S100A2. Forty-eight hours after transfection, we collected the cells and used a Total Protein Extraction Kit for further acquisition of cell lysate. Then, we added loading sample buffer to the lysate after clarifying it with centrifugation (at 14,000 rpm, 10 min), heated for 5 min at 95°C, and separated by 10% sodium dodecyl sulfate-polyacrylamide before transferring it to a nitrocellulose membrane. Nitrocellulose filters were blocked by 5% non-fat milk, and then incubated with primary antibody against S100A2 (PAC009Hu01, USCN Life Sciences Inc., Wuhan, China, 1/500 dilution) at 4°C overnight. At the same time, the mouse GAPDH antibody was set as an endogenous control. Subsequently, we rinsed the membrane, added the secondary antibodies (SAA544Rb59, USCN Life Sciences Inc., Wuhan, China, 1/5000 dilution) with horseradish peroxidase-labeled polymer to it, re-rinse, and then used enhanced chemiluminescence to observe the imaging.
MTT proliferation growth curve
The MTT assay was used to analyze cell proliferation. Firstly, the three group cells (Calu-6, Calu-6/neo, Calu-6/S100A2) at logarithmic phase were collected, centrifuged to cell suspension, and adjusted to a concentration of 5–10 × 104/ml. Then, 100 µl cell suspension was seeded into every well of 96-well plates. After being cultured in incubator, we measured the absorbance at 12, 24, 48, 72 and 96 hours respectively. Next, a total of 10 µl MTT stock solutions was added to every well, then incubated at 37°C for 4 hours before adding Formazan solution. Finally, the absorbance of each well was detected by ELISA at 490 nm.
Transwell migration and invasion assay
To examine cell migration in vitro, we applied the transwell assay. Logarithmic cells were taken, digested by 0.25% trypsin, centrifuged at 800 rpm for 5 min, and adjusted to a density of 1*106/ml. We placed the Calu-6, Calu-6/neo, Calu-6/S100A2 cells (each 100 µl) in the upper chamber of six-well transwell plates, which was matrigel-uncoated and consisted of 8.0 µm pore size filters, and added 600 µl DMEM medium (containing 30% FBS) to the lower chamber. The plates then were incubated under the condition of 37°C and 5% CO2 for 48 hours. After the incubation, we removed the upper cells with cotton-tipped swabs, fixed the filters in ethanol and stained it with hematoxylin. Five separate fields of cells on the underside were observed and took photography under the 100-fold optical microscope. The procedures of transwell assay for cell invasion detection were similar as above processes. The slight difference was that six-well transwell plates used was matrigel-coated.
Flow cytometry
We applied flow cytometry to investigate S100A2’s function in cell apoptosis and cell cycle. Cell suspension was prepared by centrifugation of logarithmic phase cells, and the count was adjusted to 3*105/mL. Approximately 2000 µl cell suspension was placed into 6-well plate, incubated for 48 hours, and then washed twice by cool PBS, centrifuged at 1000 rpm for 5 minutes at 4°C. Next, we collected a number of 1–5*105 cells, mixed them with 100 µl binding buffer after absorbing PBS. 10 µl Annexin V-FITC and 5 µl of PI staining solution were added afterwards before the incubation at room temperature for 15 min in the dark. At the last step, the mixture was added to 400 µl binding buffer and analyzed by FACSCalibur system in one hour. As for the flow cytometry procedures for cell cycle, after the centrifugation, cells were fixed in 75% ethanol for 1 h, washed by PBS, treated with 100 L RNaseA solution, and then resuspended before being bathed in water at 37°C for 30 minutes. The cell precipitation was incubated in the dark at 4°C for 30 minutes in the presence of 400 µl PI dyeing solution, and the mixture was then analyzed by flow cytometry at an excitation wavelength of 488 nm. We used Cell Quest and MidFit softwares to determine the cell cycles, which mainly contains three major phases - G1, S, G2.
Statistical analysis
GraphPad Prism 7.0 and the SPSS 22.0 statistical package were performed to make bar graphs and analyze data. A two-tailed P < 0.05 was considered to indicate statistical significance.