Cell lines and culture
Human esophageal cancer cell (Eca109) was purchased from the American Type Culture Collection (Manassas, USA). Eca109 cell maintained in RPMI 1640 medium (Gibco, USA) containing with 10% feta bovine serum (Gibco, USA) and 1% penicillin streptomycin (Gbico, USA) in an atmosphere of 5% CO2 at 37 °C.
Cell viability assay
The changes of cell viability were evaluated by employing the Cell Counting Kit-8 (CCK-8) assay. Cells (2.5 × 103) per well were cultured in 96-well plate overnight. To investigate the proliferation effect of Ginsenoside CK on Eca109 cell, cells were maintained with ginsenoside CK for 72 h. Similarly, lentivirus transfection cells were maintained in 96-well plate for 24 h, 48 h, 72 h or 96 h, then added with 10ul CCK-8 reagent (New Cell & Molecular Biotech, China) to each well, and then incubated for 2h at 37 ◦C. The absorbance value (OD450) was detected by using the microplate reader (Bio-Tek, USA).
VEGF-A knockdown cell line
Eca109 cells were transfected with knockdown lentivirus sh-VEGF-A, and the corresponding negative control lentivirus sh-NCwhich were purchased from Hanbio (Shanghai, China). Puromycin was used to screen the stably transfected cells. Western blot analysis was applied to evaluated the efficiency of lentivirus transfection.
Wound healing assay
Eca109 cell (5×105) were seeded in 6-well plates for 24h and scraped by a sterile pipette tip. Cells were cultured with DMSO or ginsenoside CK in FBS-free medium. sh-NC and sh-VEGF-A cells were maintained with FBS-free medium. The Zen Imaging software (Carl Zeiss, Germany) was applied to observe images at 0h, 24h and 48h. The Image J software (USA) was employed to calculate the scratch area.
Cell migration and invasion assays
The migration and invasion assays were employed by transwell chamber (BD, USA) with the presence of Matrigel (BD, USA) for invasion, and absence of Matrigel for migration. Cells (5x104) per well pretreat with Ginsenoside CK or lentivirus were planted into the upper chamber with 100 ml serum-free medium, and the lower chamber with 600 ml complete medium. After incubation at 37℃ for 24 h, the lower chamber cells were fixed with 70% methanol and then stained by crystal violent (Beyotime, China). The migrated or invaded cells were counted.
Ec109 cells (1.5 × 105) were maintained in 6-well plates at overnight and treated with ginsenoside CK or DMSO for 48 h. sh-NC and sh-VEGF-A cells (1.5 × 105) were planted in 6-well plates for 48 h. The rates of apoptosis cells were assessed by applying the Annexin V-FITC Apoptosis Detection Kit (Beyotime). The results were detected by the CytoFLEX flow cytometer (Beckman).
Western blot analysis
Membrane and Cytosol Protein Extraction Kit (Beyotime, China) and Bicinchoninic Acid Protein Assay Kit (Beyotime) were used for protein extracted and quantified. Proteins were separated on 10% SDS polyacrylamide gel (Beyotime) and then blotted onto PVDF membranes (Millipore, USA). The PVDF membranes were incubated in 5% skim milk for 2h. After that, all membranes were cultured with the primary antibodies of anti-Tubulin (Affinity, USA), anti-VEGF-A (Affinity), anti-Pi3k (Affinity), anti-P-Pi3k (Affinity), anti-Akt (Affinity) and anti-P-Akt (Affinity) overnight at 4 °C. The membranes were then incubated with corresponding secondary antibody (Affinity) for 1h. The protein bands were finally detected by chemiluminescence detection system (ProteinSimple, USA).
SPSS 24.0 software (SPSS Inc., Chicago, USA) was applied to data analysis, presented as mean ±SD. The difference between two groups were analyzed by Student’s t-test. P value less than 0.05 was defined as statistically significant.