Paxilline br Materials and methods br Clinical samples br Si
2. Materials and methods
2.1. Clinical samples
Sixty clinical specimens including TNBC and matched tumor-ad-jacent tissues were obtained from patients, who underwent modified radical mastectomy for breast cancer. Patients who received pre-operative immunotherapy, chemotherapy or radiotherapy were ex-cluded. Prior to the collection and use of clinical specimens, each pa-tient signed an informed consent form. The agreement on clinical specimens involved in this study was licensed by the research ethics committee of The First Aﬃliated Hospital of Bengbu Medical College.
2.2. Immunohistochemistry (IHC)
TNBC tissues were cut into 4 μm slices, and embedded in paraﬃn. Later on, sections were incubated with VEGFR-2 primary antibody (ab2349, Abcam, Cambridge, MA, USA) overnight at 4 °C. Then, the slides were incubated with peroxidase combined secondary antibody (ab7090, Abcam). Then, DAB solution was performed according to the manufacturer’s specification. VEGFR-2 staining was evaluated under a light microscope at 400 magnification. Staining intensity was scored manually: no staining = 0, weak staining = 1, moderate staining = 2, and strong staining = 3. Tumor Paxilline in five fields were randomly se-lected and scored to get the average percentage of positively stained cells (0–100%). 0–5% scored 0; 6%–35% scored 1; 36%–70% scored 2; more than 70% scored 3. The final IHC score was designated as low or high expression group using the percent of positive cell score × staining intensity score as follows: low expression was defined as a total score < 4 and high expression with a total score ≥4.
Human TNBC cell line MDA-MB-231 was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). MDA-MB-231 cells were maintained in Dulbecco’s modified Eagle’s medium (DEME, HyClone, Logan, UT, USA) supplemented with 10% fatal bovine serum (FBS, HyClone, Logan, UT, USA) at 37 °C in a humidified incubator with 5% CO2.
2.6. Flow cytometric analysis of cell apoptosis
MDA-MB-231 cells were plated onto 6-well plates overnight and treated with cisplatin (5 μM) or/and apatinib (10 μM) for 72 h. The harvested cells were fixed in ethanol (70%, v/v) and washed twice with ice-cold PBS. After that, 5 μL FITC-conjugated annexin V and 5 μL propidium (PI) were added in the situation. Then, incubated for 15 min in the dark at room temperature. Following incubation, the cells were measured using flow-cytometer (BD, Franklin Lake, NJ, USA) with WinMDI 2.9 software.
2.7. Western blotting
MDA-MB-231 cells were plated onto 6-well plates overnight and treated with cisplatin (5 μM) or/and apatinib (10 μM) for 72 h. Proteins concentration were detected by Bradford Protein Assay Kit (Beyotime, Shanghai, China). Then, equivalent amounts of proteins were separated using 10% SDS-PAGE electrophoresis. Later on, the gels were trans-ferred onto PVDF membranes (Thermo Fisher Scientific) in 2 h. After that, the PVDF membranes were blocked with 5% defatted milk in TBST at room temperature for 1 h. Then the membranes were incubated with primary antibodies overnight: anti-Bax (Abcam; ab32503) (1:1000), anti-active caspase 3 (Abcam; ab2302) (1:1000), anti-Bcl-2 (Abcam; ab196459) (1:1000), anti-β-actin (Abcam; ab8227) (1:1000), anti-p-VEGR2 (Abcam; ab5473) (1:1000), anti-p-Akt (Abcam; ab38449) (1:1000), anti-p-mTOR (Abcam; ab84400) (1:1000). After washing, the membranes were incubated with secondary antibody (1:5000, Abcam; ab7090) at room temperature for 1 h. Finally, the PVDF membranes were incubated with electro chemiluminescence (ECL) reagent (Santa Cruz Biotechnology) to measure the chemiluminescence intensity. The relative expression of protein was quantified by normalizing to β-actin.
2.8. Transwell migration and invasion assays
For migration assay, MDA-MB-231 cells were incubated in serum-free DMEM for 24 h. Cells (2.5 × 104 cells) were then seeded onto fil-ters (8 mm pore size) in transwell inserts (Corning, New York, NY, USA) in filter and treated with cisplatin (5 μM) or/and apatinib (10 μM) for 72 h. The lower chamber of each well was added with DMEM con-taining with 10% FBS. After incubation, cells on the upper surface were removed with a cotton swab, and cells that had migrated to the lower surface stained with a solution containing 50% isopropanol and 0.5% crystal violet. The numbers of migrated cells at least 5 random fields from each membrane was counted. For invasion assay, identical methods were performed as those described in the migration assay except that the upper chamber is pre-treated with 100 μL of Matrigel.
2.9. Statistical analysis
The SPSS19.0 software (Chicago, IL, USA) was used for statistical analyses. Data were analyzed by one-way analysis of variance with the Bonferroni multiple comparison test. Comparison between two groups was performed using Student’s t-tests. Data were represented as mean ± S.D. P < 0.05 was regarded as statistically very significant.