Cell culture and lentiviral transfection
LX-2 cells (DSMZ CVCL_5792) purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). The ERβ-overexpressing vector pHBLV-CMV-MCS-3FLAG-EF1- ZsGreen-T2A-PURO and the empty vector were purchased from HANBIO (Shanghai, China). LX-2 cells were cultured in DMEM (Hyclone, USA) with 10% fetal bovine serum (FBS, Ever Green, China) and were maintained in a humidified incubator at 37 °C with 5% CO2. Lentivirus transfection was operated according to the manufacturer’s instructions. For cell transfection, LX-2 cells were transferred into 6-well plates at the density of 2 × 105 cells/well. After overnight culturing, LX-2 cells were transfected with ERβ overexpressing lentivirus for 48 h or 72 h. To determine the optimal multiplicity of infection (MOI), the lentivirus with MOI of 10, 20, 30 and 40 was respectively added to each well according to the instructions. The expression of green fluorescent protein in the transfected cells was observed by fluorescence microscope (Olympus, Japan) 24 h and 48 h after transfection. Stable transfected LX-2 cells were screened using fresh medium containing 2 μg/mL purinomycin for 24 h. Overexpression of ERβ was identified by determination of mRNA and protein expression.
LX-2 cell proliferation assay
The effects of calycosin (Phytomarker Ltd., Tianjing, China) on cell proliferation were evaluated by the cell counting kit-8 (CCK-8) assay (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Quiescent LX-2 cells were first treated with calycosin (25, 50, 100, 200, 300, 600 μM) to investigate the cytotoxicity and safe dose range of calycosin. Then, LX-2 cells were pretreated with safe concentrations of calycosin (25, 50, 100, 200 μM) or E2 (1μM, Shanghai yuanye biotechnology Co., Ltd., Shanghai, China) for 12 h and then stimulated with 10 ng/ml TGF-β1 (R&D Systems, Minneapolis, MN, USA) for 12 h.
LX-2 cells or transfected cells (MOI = 40) were seeded into 96-well plates at a density of 5 × 103 cells/well at 24 h, 48 h and 72 h. TGF-β1 (10 ng/ml) was added into cells (transfected for 72 h) for another 12 h to show the effect of TGF-β1 on proliferation of ERβ overexpressing cells. After incubated with serum-free media for 24 h, LX-2 cells or transfected cells were cultured in DMEM containing calycosin dissolved in dimethyl sulfoxide (DMSO, Beyotime, Shanghai, China) or E2 for 12 h. TGF-β1 with final concentration of 10 ng/ml was then added for another 12 h. At the end of the treatment, the medium was replaced with 90 μl DMEM supplemented with 10% FBS, and 10 μl CCK-8 was added into each well, then cells were incubated for an additional 3 h. Absorbance was detected by a Multiskan GO (Thermo, USA) at 450 nm.
Cell migration assay
According to our previous method , the migration ability of LX-2 and transfected cells was inspected by 24-well corning transwell chambers (8.0 μm pore size polycarbonate membrane, Corning Costar, USA). Cells were washed with PBS after digestion and resuspended in serum-free DMEM. Then 2 × 104 cells were seeded into the upper chambers and 0.6 ml DMEM containing 10% FBS was added to the bottom chambers. After 12 h culturing, cells were treated with calycosin (200 μM) or E2 (1μM) for 24 h, and then 10 ng/ml TGF-β1 was added to upper chambers. After 12 h of incubation, all unmigrated cells were removed from the upper surface of the filters and the migrated cells were fixed and stained with crystal violet solution (Shanghai yuanye Bio-Technology, Shanghai, China). All the stained cells were counted in five different fields under a 400-fold microscope (Olympus, Japan).
According to our previous method , LX-2 cells (2.5 × 104 cells/well) with or without ERβ overexpression were seeded onto the cover glass on a 24-well cell culture plate. After starving overnight in DMEM containing 2% FBS, except cells in control and vechile groups, all cells were stimulated by 10 ng/ml TGF-β1 for 12 h with or without pretreatment of calycosin (200 μM) or E2 (1μM) for 24 h. Then cells were washed with PBS three times and fixed in 4% paraformaldehyde (Beyotime, Shanghai, China) for 15 min at room temperature. After twice washes, cells were incubated for 10 min with PBS containing 0.2% Triton X-100 (Sangon Biotech, Shanghai, China). Cells were washed by PBS twice again and then blocked in PBS containing 5% BSA for 1 h at room temperature. Afterwards, the cells were incubated overnight at 4 °C with anti-α-SMA (1:300) primary antibody (Bioss, China) and then gently washed with PBS 3 times and further incubated with TRITC-conjugated secondary antibodies (1:50, ZSGB-BIO, China) for 1 h at room temperature in dark. Finally, cells were washed and then incubated with DAPI (1 μg/mL, Beyotime, Shanghai, China) for another 15 min at room temperature. The co-localization expression was visualized under confocal microscope (Nikon A1, Japan).
Quantitative real-time PCR
Quantitative real-time PCR (qRT-PCR) was carried out according to our previous methods . Total RNA extracted from cells were lysed by TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Then the synthesis of cDNA was performed using the primeScript™ RT reagent kit (Takara Biotechnology, Shiga, Japan) and quantitative PCR was performed on a 7500 Real Time PCR system (Applied Biosystems, Foster City, CA, USA) with SYBR® premix Ex Taq™ (Takara, Cambridge, MA, USA) according to the instructions. Targeted cDNAs were amplified for 95 °C, 30 s (1 cycle); 95 °C, 5 s; and 60 °C, 34 s (40 cycles). The primers used for PCR amplification were as follows: ERβ (forward 5′-GAATGGTCAAGTGTGGATCCAGGAG-3′; reverse 5′-CTCCATCCAGCAGTT-TCCAAGAGG-3′) and β-actin (forward 5′-TCCTCCTGAGCGCAAGTACTCT-3′; reverse 5′-GCTCAGTAACAGTCCGCCTAGAA-3′). The relative level of ERβ mRNA was quantified by the comparative threshold cycle (ΔΔCt) method.
Western blotting analysis
Quiescent LX-2 cells were pretreated with calycosin (200 μM) or E2 (1μM) for 24 h and then TGF-β1 (10 ng/ml) was added for 12 h. LX-2 cells were washed twice with cold PBS and lysed in RIPA (Beyotime, Shanghai, China) containing 1% PMSF (Beyotime, Shanghai, China) and 1% phosphatase inhibitor (New Cell& Molecular Biotech, Suzhou, China) for 30 min on ice. Cells were scraped from the plate and centrifuged at 12000 r/min for 30 min at 4 °C. The protein concentrations were measured by BCA assay kit (Beyotime, China) according to the manufacturer’s protocol. Then samples were electrophoresed by 10% SDS-PAGE gels and subsequently transferred to 0.45 μm PVDF membranes (Millipore, USA) at 200 mA for 1 h. The membranes were blocked with 5% nonfat dried milk (Beyotime, Shanghai, China) in Tris-buffered solution (TBST, pH 7.6, 0.05% Tween 20) at room temperature for 1 h. Then the membranes were washed with TBST 3 times for 10 min each. According to the position indicated by marker, the membranes with target proteins were cut off, and thus the images with adequate length were not obtained. The membranes were incubated separately at 4 °C overnight in the following diluted primary antibodies: mouse anti-ERβ antibody (1:1000, Abcam, UK), rabbit anti-α-SMA antibody (1:1000, Bioss, China), rabbit anti-Col I antibody (1:1000, Bioss, China), rabbit anti-MMP1 (1:1000, Bioss, China), rabbit anti-TIMP1 (1:1000, Bioss, China), mouse anti-JAK2 antibody (1:1000, Bioss, China), rabbit anti-p-JAK2 antibody (1:1000, Affinity, America), rabbit anti-STAT3 antibody (1:1000, Bioss, China), rabbit anti-p-STAT3 antibody (1:1000, Bioss, China), rabbit anti-GAPDH antibody (1:2000, Bioss, China), mouse anti-β-actin antibody (1:1000, ZSGB-BIO, China). After 3 washes, membranes were incubated in diluted secondary antibodies (anti-mouse IgG-HRP 1:10000, anti-rabbit IgG-HRP 1:10000, ZSGB-BIO, China) at room temperature for 1 h. The proteins on the PVDF membranes were detected with electrochemiluminescence (ECL) western blot detection reagents (Bridgen, China). Image J was used to analyze the grayscale of stripes.
Data were represented as mean ± SD for at least three independent experiments. The data were analyzed by Student t test for paired comparisons using SPSS 23.0 and Graphpad prism 7.0 software. P < 0.05 was considered statistically significant. The significance between groups was marked on the graphs.