Main reagents
The antibodies against E-cadherin (#562869) and N-cadherin (#561553) were purchased from BD Biosciences (San Jose, CA, USA) , and antibodies against Vimentin (#5741), β-catenin (#8480), GAPDH (#5174) and HRP-linked anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (Danvers, MA, USA). Fetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, USA) and DMEM medium were purchased from Hyclone (China). Polyvinylidenefluoride (PVDF) membrane was purchased from Merck Millipore (USA). ECL substrate kit was purchased from Thermo Scientific (USA). TGF-β1 (80116) was purchased from Sino Biological Inc. (China). RIPA lysis buffer and BCA Protein Assay Kit were purchased from Beijing Solarbio Science & technology Co., Ltd. RNA extraction kit, cDNA synthesis kit and PCR kit were purchased from TaKaRa (Japan).
Cell lines and culture conditions
Rat VSMCs were donated by the Department of Pathophysiology at Nanjing Medical University. The cells were cultured in the complete medium at 37 ℃ in the 5 % CO2 incubator. The cells were digested with 0.25 % trypsin and passaged. Well-grown cells within 10 generations were collected for subsequent experiments.
Cell grouping
Experimental cells were divided into TGF-β1 induction group with different concentrations, in which the control group used complete medium without TGF-β1, the low concentration group with 2 ng/ml TGF-β1 complete medium, the medium group with 4 ng/ml TGF-β1 complete medium, and the high group with 8 ng/ml TGF-β1 complete medium, all cultured for 48 h at different concentrations.
Wound healing assay
Rat VSMCs were inoculated in 6-well plates and cultured with DMEM containing 2 % serum. When the cell growth density reaches 80 %-90 % fusion, 200 μl pipettes were used to scratch the bottom of the orifice plate. The scratched cells were cultured in an incubator of 5 % CO2 at 37 ℃, and the cell scratch healing was photographed at 0 h, 24 h and 48 h respectively. The average scratch width was calculated to reflect the extent of the scratches merging. Each experiment was repeated three times.
Transwell assay
The suspension of rat VSMCs was inoculated to a 6-well plate at 2 ml per well. The cells were further cultured for 12 h and digested with trypsin. The cells were counted and the cell concentration was adjusted to 2 × 105/ ml. Then 200 μl of cell suspension diluted by serum-free DMEM was transferred to the upper chamber, while 600 μl of complete medium was added into each well of the lower chamber. After further culture for 24 h, the cells were fixed in formaldehyde and stained with crystal violet. Cells in the upper chamber were wiped off. Cells migrating to the lower chambers were counted under a microscope. Three visual fields were randomly selected for statistical analysis in each chamber. Each test was done in triplicate.
Western blotting
Detection was performed using RIPA lysis buffer in strict accordance with the manufacturer's manual. The cells were cultured in 6-well plates, and when the cell density reached 80 %-90 %, and 200 μl of RIPA lysis buffer containing 10 % protease inhibitor was added. After sufficient lysis on ice for 10min, the cells were centrifuged at 4 ℃ and 12000 × g for 10min, and the supernatant was collected. Concentrations of each target protein were detected by using BCA protein assay kit. The protein samples were properly mixed with 5 × protein loading buffer. The protein was sufficiently denatured by boiling at 100 ℃ for 10min and then preserved at -20 ℃. For each well the loading amount of total protein was 60 μg. 10 % polyacrylamide gel electrophoresis was performed, and the proteins were electrotransferred to the PVDF membrane, which was sealed with 5 % defatted milk powder for 2 h. The cells were incubated with E-cadherin antibody (1:1000), N-cadherin antibody (1:1000), Vimentin antibody (1:1000), β-catenin antibody (1:1000) and GAPDH antibody (1:3000) at 4 ℃ overnight, respectively. On the next day, PBS containing 0.1 % Tween-20 (PBST) was used to washing the cells for three times. This was followed by incubation with corresponding HRP-conjugated secondary antibodies at the room temperature for 1 h. The membrane was washed with PBST for three times, and the grayscale values of each band were analyzed using the ChemiDoc XRS+ imaging system. GAPDH was used as internal reference. The grayscale value ratio of each target protein to internal reference was defined as the relative expression of target protein. Each test was done in triplicate.
RT-qPCR
Primers were designed for each target gene and internal reference gene GAPDH using Primer Premier 5.0. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Primer sequences were as follows: N-cadherin forward, 5'-CAGTGTACAGAAT
CAGTG-3' and reverse, 5'-CAACAGTAAGGACAAACA-3'; E-cadherin forward, 5'- CAAGTGAC CACCTTAGAG-3' and reverse, 5'-GAATTTGCAATCCTGCTT-3'; β-catenin forward, 5'-ACCTATACTTACGAAAAACTAC-3' and reverse, 5'-CCACCAGCTTCTACAATA-3'; Vimentin forward, 5'-GACGCCATCAACACCGAGTT-3' and reverse, 5'-CTTTGTCGTTGGTTAGCTGGT-3' and GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-TGGTGAAGACGCCAGTGGA-3′. Total RNA extraction was performed from the cells using total RNA extraction kit, and cDNA was synthesized by using PrimeScriptTM RT Master Mix (TaKaRa, Dalian, China), followed by the addition of SYBR Green on an ABI 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). Amplification curve and dissolution curve were plotted and CT values of each target gene were calculated. GAPDH was used as internal reference. The relative expression of mRNA of each target gene was calculated using 2 -ΔΔCt method. Each test was done in triplicate.
Statistical analysis
Statistical analysis was performerd using SPSS 20.0 software. Measurements were expressed as x±s. Inter-group comparison was conducted by one-way ANOVA, and pairwise comparison by q-test. P < 0.05 indicated significant difference.