The expression plasmids pcDNA3.1-NFAT2 and its empty vector pcDNA3.1 were purchased from promega (WI, USA). The Lipofectamine 2000 was obtained from Invitrogen (Thermofisher Scientific., USA). Pluronic F-127 was bought from Genecopoeia (MA, USA). Fluo-4/AM, anhydrous dimethyl sulfoxide (DMSO) and cell counting kit-8 (CCK-8) were acquired from Dojindo Molecular Technologies Inc. (Tokyo, Japan). Antibodies against NFAT2 (RRID: AB_2152507), Egr2 (RRID:AB_1139730), FasL (RRID:AB_302235), ERK (RRID:AB_11157324) and β-actin (RRID:AB_764434) were obtained from Abcam (Cambridge, UK). Antibodies against AKT (RRID:AB_329827), p-AKT(Try326) (RRID:AB_1264114), p-ERK(S189) (RRID:AB_490903), c-myc (RRID:AB_2151827) and Cox-2 (RRID:AB_2084968) were bought from Cell Signaling Technology (MA, USA). The HRP-conjugated secondary antibody was acquired from Bioss, Inc. (Beijing, China). Annexin V-FITC apoptosis detection kit was obtained from BD Biosciences (NJ, USA). The Click-iT Plus 5-ethynyl-2’-deoxyuridine (EdU) Flow Cytometry Assay kit was bought from Invitrogen. All chemical reagents used in this study were of analytical grade and all reagents for cell culture were purchased from Gibco BRL Life Technologies (MD, USA).
2.2 Cell culture and tissue samples
Human liver hepatocellular carcinoma (HepG2) cells (RRID:CVCL_0027) which were bought from American Type Culture Collection (ATCC, Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) including 10%(V/V) fetal bovine serum (FBS), 0.1 mg/mL streptomycin and 100 U/mL penicillin in a 37 °C humidified incubator with 5% CO2. Cells were fed every day and were sub-cultured once they reached 90% confluency.
Hepatic carcinoma samples, including carcinoma samples (CS) and adjacent nontumor samples (AS) were obtained from 20 patients who were diagnosed as HCC and received hepatectomy during 2018 at the Tianjin First Central Hospital. The age of those patients ranged from 20 to 78 years. The tissue samples were stored at liquid nitrogen for PCR and western blot assay.
2.3 Transient transfection assay
HepG2 cells were cultured in 6-well plate with complete medium for 24 h. Then, the cells were respectively transfected with 70 nmol/L plasmids pcDNA3.1-NFAT2 and the control vector, pcDNA3.1 in fresh serum free medium for 24 h. Lipofectamine 2000 was used in the transfection according to the manufacturer’s instructions. All the cells stably transfected with pcDNA3.1-NFAT2 were confirmed by western blot and RT-qPCR analysis and designated as HepG2/NFAT2. The cells stably transfected with pcDNA3.1 vector were regarded as negative control (NC). The HepG2/NFAT2 and NC cells were sub-cultured and applied for the following analysis.
2.4 Cell counting kit-8 (CCK-8) assay
The CCK-8 assay was performed to detect the viability of HepG2/NFAT2 and NC cells. The cells, in brief, were seeded into 96-well plate and were cultured in 200 μL complete medium for 12 h, 24 h, 48 h and 72h, respectively. Then, CCK-8 reagents were respectively added to the medium of corresponding wells and the cells were incubated at 37 °C for another 2 h. Subsequently, the absorbance of each well was monitored at optical density (OD) 450 nm by Microplate Reader (Bio-Tek Instruments, VT, USA).
2.5 Annexin V-FITC staining and EdU labeling assay
The cells were seeded in a 6-well plate at a density of 2 ᵡ 105 cells/well, with the complete medium overnight. The cells were collected, washed by ice-cold PBS for three times and then resuspended by 1×binding buffer. 100 μL cell suspension was stained by 1/1000 (v/v) Annexin V-FITC for 20 min at room temperature, in the dark. Next, the cells were washed thoroughly by PBS and stained with 2 μg/mL propidine iodide (PI) in deliquated binding buffer. The apoptosis evaluation was performed by flow cytometer (Beckman Coulter, CA, USA) and analyzed by FlowJo software (RRID:SCR_008520) (Tree Star, OR, USA).
To assess the cell proliferation, the EdU (5-ethynyl-2’-deoxyuridine) labeling assay was performed by incubating NC and HepG2/NFAT2 cells with serum free medium containing 10 μmol/L EdU for 2 h. Then, the cells were washed by PBS and fixed for 15 min at room temperature. The fixed cells were washed and re-suspended in permeabilization buffer for 10 min in dark place. The cell suspension was incubated in Click-iT plus reaction cocktail for 30 min at room temperature in the dark. The stained cells were washed and analyzed by flow cytometer.
2.6 Transwell assay
To evaluate the invasion efficiency, the transwell assays were conducted in 24-well transwell inserts (8.0 μm pores, BD Biosciences) according to the recommendations by the manufacturer. Briefly, cells (1ᵡ105 cells/chamber) were plated in the upper chamber, with serum free medium while medium containing 10% FBS, using as chemoattractant, was placed in the lower chamber. After 24 h incubation, migrated cells in the bottom surface of the upper chamber membranes were fixed by methanol for 5 min and then stained with 0.1% crystal violet in 20% ethanol for 15 min. The random visual fields were imaged by a light microscope. The values of invasion were expressed with the mean absorbance of OD 570 nm per assay.
2.7 Wound healing assay
The wound healing assays were applied for assessing the migration capacity of cells. Cells were seeded into 6-well plates with complete medium, at a density of 5ᵡ105 cells/well and then cultured for 24 h. The linear wounds were scratched by a pipette tip on the completely confluent monolayer cells, timed 0 h and then cells were cultured in serum free medium for 24 h, the last moment timed 24 h. The images at 0 h and 24 h were captured by an inverted microscope and the migration % = (the distance of two sides at 0 h (D0) – the distance of 24 h (D24))/D0.
2.8 Fluo-4-Ca2+ complex analysis
HepG2 cells were cultured in a 6-well plate for 24 h and then incubated by hank’s balanced salt solution (HBSS, without Ca2+ and Mg2+) containing 1 μmol/L fluo-4/AM and 0.5‰ pluronic F-127 for 20 min at incubator. After fluo-4/AM loaded, the cells were washed by HBSS for three times and then equilibrated in HBSS for 30 min in the dark. The fluorescence intensity of fluo-4-Ca2+ complex was detected by a Live Cell Imaging System (AF7000, Leica, Germany) with Ex 488 nm and Em 516 nm, and 20 mmol/L KCl was added into wells at almost 15 s. The fluorescence curve was calculated by ∆F/F0 = (the mean fluorescence intensity of cells – the background intensity)/the background intensity.
Total RNAs in cells and tissues were extracted by TRIzolTM Plus RNA purification kit (Invitrogen) and first-strand cDNA was synthesized by the ImProm-Ⅱ reverse transcription system (Promega). Real-time PCR assays were conducted by the SYBR Green Mastermix (Applied Biosystems) and all procedures were performed according to the manufacturer’s instructions. Specific primers used for genes were as follows: NFAT2 forward, 5’-GCTATGCATCCTCCAACGTC-3’; and reverse, 5’-AGTTFFACTCGTAGGAGGAG-3’; EGR2 forward, 5’-TCAGCATCTCCCAACCTAT-3’; and reverse, 5’-ACAACAAACACTACCACCCT-3’; FASL forward, 5’-GTTCTGGTTGCCTTGGTAG-3’; and reverse, 5’-CATCTGGCTGGTAGACTCT-3’; COX-2 forward, 5’-GAAAGCCCTCTACCATGACATC-3’; and reverse, 5’-CACCCTTTCACATTATTGCAGA-3’; c-myc forward, 5’-GCCACGTCTCCACACATCAG-3’; and reverse, 5’-TCTTGGCAGCAGGATAGTCCTT-3’; β-actin forward, 5’-CTGGGACGACATGGAGAAAA-3’; and reverse, 5’-AAGGAAGGCTGGAAGAGTGC-3’. All data were analyzed by the ABI7300 system SDS software (Applied Biosystems) and the mRNA relative expression was calculated by the 2-∆∆Ct method. The β-actin expression was regarded as control, so the expression value for each gene was normalized to β-actin expression in each sample.
2.10 Cell treatment
The cells were seeded in a 6-well plate at a density of 2 ᵡ 105 cells/well, with the complete medium overnight. 1 ᵡ 10-3 mol/L ionomycin and 1 mg/mL 12-myristate 13-acetate (PMA) were applied to pretreat cells for 8 h. The cells were then washed and subjected to western blot analysis.
2.11 Western blot
Total proteins in cells were extracted by ice cold RIPA lysis buffer added PMSF for 30 min. While the process of hepatic protein extraction was performed by firstly pulverizing hepatic tissues in liquid nitrogen and lysing the fragments in ice cold RIPA buffer for 30 min. The protein extractions were centrifugated at 15000 rpm at 4 °C for 10 min and the supernatants were quantified by bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The equivalent protein samples were subjected to 10% SDS-PAGE followed by western blot analysis. The PVDF membranes were blocked by 5% skim milk in TBST for 1 h at room temperature, and next were incubated overnight at 4 °C by the primary antibodies diluted in TBST: anti- NFAT2 (1:1000), Egr2 (1:1000), FasL (1:2000), AKT (1:1000), p-AKT (1:1000), ERK (1:1000), p-ERK (1:1000), COX-2 (1:1000), c-myc (1:2000), and β-Actin (1:1000). After primary antibodies incubation, the PVDF membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. The proteins were stained with ImmobilonTM Western Chemiluminescent HRP Substrate detection reagent (Millipore, MA, USA) and imaged using Image LabTM software (Bio-Rad, VA, USA). To quantify the relative intensity of proteins, the ratios of target proteins to β-actin signal were calculated by ImageJ software. The full-length bots/gels are presented in the Supplementary Figure 2.
2.12 Immunohistochemistry (IHC)
Paraffin sections were deparaffinized in xylene and then successively rehydrated by graded alcohol. Antigen retrieval was conducted through boiling the section samples in EDTA (pH 6.0) in a microwave oven at 95 °C for 10 min. The section samples were incubated in 3% hydrogen peroxide for 30 min at room temperature and then blocked by 20% goat serum for 40 min. Section samples were incubated with anti- NFAT2 antibody in 20% goat serum (1:100) overnight at 4 °C. All sections were incubated by HRP-conjugated secondary antibody for 60 min at room temperature. At last, the protein was stained by diaminobenzidine and the sections were counterstained with hematoxylin.
2.13 Statistical analysis
Statistical analysis was performed using SPSS 24.0 (RRID:SCR_002865) (SPSS Inc., Chicago, USA) and all values are presented as the mean ± standard deviation (SD). A two-tailed, student’s t-test was used to statistical comparison between two groups. For data that didn’t distribute normally, a paired two-tailed Wilcoxon matched-pairs signed-rank test or a two-tailed unpaired Mann-Whitney test were applied to calculate its significance. The p values less than 0.05 was regarded as statistically significant.