Cell culture
Human breast adenocarcinoma cell line SK-BR-3 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) under 5% CO2 at 37°C.
MTT assay
1×104 cells per well in 96-well plates were incubated for 24 hours. After removing the culture medium, 100 μL medium containing 0.5 mg/ml MTT solution (Roche Diagnostics Deutschland GmbH,Basel,Switzerland,11465007001) was added, followed by 37°C incubation for 4 hours. Then, after removing MTT solution-contained medium, 150 μL DMSO per well was added, followed by shaking for 10 minutes in dark. A570 nm microplate reader was used to examine OD values.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was extracted from SK-BR-3 cells by Trizol reagent, followed by reverse transcription by Prime Script™ RT enzyme. SYBR Premix Ex Taq™ enzyme was used to perform RT-PCR according to the manufacturer's instruction (Takara Bio,Kyoto, Japan,RR037A). The primer sequences included: Human GAPDH primers: forword primer 5’-CCACTCCTCCACCTTTG-3’, reverse primer 5’-CACCACCCTGTTGCTGT-3’; Human PCNA primers: forward primer 5’-CCTGCTGGGATATTAGCTCCA-3’, reverse primer 5’-CAGCGGTAGGTGTCGAAGC-3’; Human Ki67 primers: forward primer 5’-ACGCCTGGTTACTATCAAAAGG-3’, reverse primer 5’-CAGACCCATTTACTTGTGTTGGA3’; Human BAX primers: forward primer 5’- TGCAGAGGATGATTGCTGAC-3’, reverse primer 5’-ACTCCAGCCACAAAGATGGT-3’; Human BCL-2 primers: forward primer 5’- CGGCTGTCGTCTGACTACAT-3’, reverse primer 5’-CCAGATCCCTCTTCTGCTTG-3’; Human MMP2 primers: forward primer 5’-TACAGGATCATTGGCTACACACC-3’, reverse primer 5’-GGTCACATCGCTCCAGACT-3’; Human MMP9 primers: forward primer 5’- TGTACCGCTATGGTTACACTCG-3’, reverse primer 5’-GGCAGGGACAGTTGCTTCT-3’.
Western blotting
Total protein was extracted from SK-BR-3 cells by RIPA lysis buffer. 1 μg/μl protein sample was used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), then protein was transfered to PVDF membrane (Millipore, Massachusetts, USA, IEVH07850). Primary antibodies were used for incubation, including anti-GAPDH (Abcam, Cambridge, UK, ab8245), anti-PCNA (Abcam, Cambridge, UK, ab29), anti-MMP2 (Abcam, Cambridge, UK, ab92536), anti-MMP9 (Abcam, Cambridge, UK, ab76003), anti-BAX (Abcam, Cambridge, UK, ab32503), anti-Bcl-2 (Abcam, Cambridge, UK, ab32124). The second antibody was used for incubation at 37°C for 1 hour. After washing, images were collected by chemiluminescent reagents and Bioshine ChemiQ4600 imaging system (Shanghai Bioshine Scientific Instrument, Co, Ltd, Shanghai, China).
Colony formation assay
200 cells per well in six-well plates were incubated at 37°C, then pipetting repeatedly and shaking gently to make the single-cell suspension. When the visible cell clone appeared at the bottom, the medium was removed, followed by washing, fixing by 4% paraformaldehyde for 20 minutes, adding 2 ml of 0.1% crystal violet staining solution for 30 minutes. After rinse, pictures were collected for cell clone counting.
Transwell invasion assay
50 μl Matrigel was added into 150 μl serum-free medium, then mixing well to add into the chamber, incubating at 37°C for 1 hour for coagulation. 3×105 cells/mL cell suspension was prepared to add 100 μl into each well. 500 μl medium containing 10% FBS was added into lower chamber, followed by incubation at 37°C for 24 hours. After washing, Matrigel and cells in the upper chamber were removed, then fixing by 4% paraformaldehyde for 20 minutes, adding 0.1% crystal violet staining solution for 15-20 minutes. After rinse, pictures were collected for invaded cell counting.
Transwell migration assay
SK-BR-3 cells were prepared with a cell suspension of 1×106 cells/ml. 100 ul cell suspension was incubated in transwell upper chamber, and the equal serum-free DMEM medium was used as the control group. 500 ul DMEM medium containing 10% FBS was added into lower chamber and SK-BR-3 cells were cultured at 37℃ in 5% CO2 for 24 hours. Filter membrane was fixed with methanol for 5 minutes, followed by staining with 0.1% crystal violet solution for 15 minutes. Cell counting was performed in different visual fields of the membrane to evaluate the migration ability of SK-BR-3 cells.
Cell wound healing assay
5×105 cells per well was added into 6-well plates for incubation at 37°C overnight. The pipette tip was perpendicular to the ruler and the horizontal line was drew on 6-well plates by scratching slowly and evenly. After washing, cells were cultured in the medium with 5% FBS at 37°C in 5% CO2. After washing at 0, 12, 24 and 48 hours, pictures were collected and cell wound healing was measured.
Flow cytometry
SK-BR-3 cells were treated by LTP for different times. For flow cytometry apoptosis analysis, cells were incubated with FITC anti-Annexin V (BioLegend) for 15 minutes, followed by PI staining. SK-BR-3 cell apoptosis was analyzed using BD FACS Verse, and data processing was conducted using FlowJo software (Ashland, OR, USA).
Statistical analysis
All experimental data were statistically analyzed using Graphpad Prism 5. The measurement data were expressed in Mean±SEM, and the comparison among multiple groups was analyzed by one-way ANOVA. The comparison between two groups was analyzed by t-test analysis. p<0.05 means statistical significance. All experimental data were obtained by at least three independent experiments.