Antibodies
Rabbit antibody against FAM96A was purchased from EpiGentek (Farmingdale, NY, USA). Rabbit antibody against FAM96B was obtained from Abcam (Cambridge, UK). Rabbit antibodies against p21, CDK4, cyclin D1, N-cadherin, E-cadherin, Slug, Snail, Vimentin, GSK-3β, p-GSK-3β (Ser9) and β-catenin were purchased from Cell Signaling Technology (CST) (Danvers, MA, USA). Rabbit antibodies against Bcl-2, Bax, Caspase 3, cyclin E, CDK2 and c-Myc were from Wanleibio (Shenyang, China). The anti-β-actin mouse antibody was from ZSGB-bio (Beijing, China). Goat anti-rabbit and goat anti-mouse HRP-conjugated secondary antibodies were from ABGENT (San Diego, CA, USA).
Cell lines and culture
Human breast cancer cell lines including MCF-7 and MDA-MB-231, and the normal breast epithelial cell line MCF-10A were obtained from Chinese Academy of Sciences. These cell lines were maintained in the DMEM medium (Hyclone, Logan, UT, USA) containing 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (HyClone) at 37 °C in a humidified cell culture incubator with 5% CO2.
Cell transfection
The specific siRNAs against FAM96A (siFAM96A), FAM96B (siFAM96B) and non-specific control siRNA (siNC) were designed and synthesized to down-regulate FAM96A and FAM96B expression by Genepharm Co., Ltd (Shanghai, China). The specific siRNA sequences targeting FAM96A and FAM96B were shown in the Supplementary Table S1. The cDNA sequences of FAM96A and FAM96B were subcloned into the pEGFP-C3 vector to up-regulate FAM96A and FAM96B expression by PCR analysis. The transfection reagent lipofectamine® 3000 (Invitrogen, Waltham, MA, USA) was used to transfect with siRNAs or recombinant plasmids into MCF-7 and MDA-MB-231 cells according to the manufacturer’s instructions. The overexpression and knockdown efficiencies of FAM96A and FAM96B in the mRNA and protein levels were detected using RT-qPCR and Western blot assays, respectively.
For specific inhibition of the Wnt/β-catenin signaling pathway, XAV-939, a potent tankyrase inhibitor that targets Wnt/β-catenin signaling, was added into the DMEM medium of the cultured breast cancer cells with a final concentration of 30 nM for 24 h (Selleck Chemicals, Houston, TX, USA).
RT-qPCR
Total RNA was extracted from the cultured cells using a RNAprep Pure Cell Kit (TIANGEN, Beijing, China), and 1 μg total RNA was reversed transcribed into cDNA with PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara, Dalian, China). RT-qPCR was used to detect the gene expression by SYBR® Premix DimerEraser Kit (Takara) according to the manufacturer’s protocols. The relative mRNA level of each gene was calculated using 2-△△Ct method and normalized by GAPDH mRNA. The specific primer sequence of each gene used in this experiment are shown in the Supplementary Table S2.
Western blot
Total proteins were extracted from the cultured cells using RIPA buffer supplemented with PMSF and cocktail protease inhibitor (Genview, Beijing, China). The concentration of each total protein sample was measured by a BCA Protein Assay Kit (Genview). Each cell sample containing 40 μg protein were subjected to separate by 10% SDS-polyacrylamide gel, and then the proteins were transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After being blocked in 5% non-fat skim milk for 2 h at room temperature, the PVDF membranes were incubated with corresponding primary antibodies at 4 °C overnight. The primary antibodies used in this experiment included anti-FAM96A, anti-FAM96B, anti-p21, anti-cyclin D1, anti-CDK4, anti-cyclin E, anti-CDK2, anti-Bax, anti-Bcl-2, anti-Caspase-3, anti-N-cadherin, anti-Vimentin, anti-E-cadherin, anti-Snail, anti-Slug, anti-β-catenin, anti-GSK-3β, anti-p-GSK-3β, and anti-c-Myc (1:500 ~ 1:1000 dilution). β-actin was served as the protein loading control (1:5000 dilution). Subsequently, the PVDF membranes were washed three times for 10 min, and then incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the protein bands were detected by ECL chemiluminescent HRP substrate (Millipore) and visualized with a Tanon 4200 chemiluminescence imaging system (Tanon, Shanghai, China).
Cell proliferation assay
Cells were seeded into a 96-well plate with 1×103 cells per well. Before daily measurement, 10 μl MTT solution (Solarbio, Beijing, China) was added into the each well of the 96-well plate at the different time points (24 h, 48 h, 72h, 96 h). After incubation for another 4 h at 37 °C, the absorbance was measured at 490 nm for the MTT solution using a microplate reader (BioTek, Winooski, VT, USA). All experiments were performed in triplicate.
Colony formation assay
Cells were seeded into a 6-well plate with 1×103 cells per well and cultured for about 14 days. After obvious colonies were formed, the culture medium was removed and the colonies were fixed with 4% paraformaldehyde (Genview) for 30 min. Subsequently, the colonies were stained with 0.1% crystal violet (Amresco, Solon, OH, USA) for 30 min. After washing and drying, the stained colonies were photographed and counted manually. The statistical results are the mean values of three independent experiments.
Cell cycle assay
Cells were harvested by trypsin digestion, washed with PBS and fixed with 70% ethanol in PBS at 4 °C overnight. Next, the fixed cells were treated with 100 μg/ml RNase A (TIANGEN) in PBS for 30 min at 37 °C, and then stained with 100 μg/ml propidium iodide (PI) solution (Solarbio) for 30 min at room temperature. Cell cycle progression was detected by FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA), and the number of cells in G0/G1, S and G2/M phase were assayed with ModFit LT 3.0 software.
Cell apoptosis assay
Annexin V-FITC/PI or Annexin V-FITC/7-AAD Apoptosis Detection Kit (Solarbio, Beijing, China) was used to evaluate cell apoptosis. In brief, the harvested cells were incubated with 5 μl Annexin V-FITC and 5 μl PI or 7-AAD in 100 μl binding buffer for 10 min according to the manufacturer’s protocols in the darkness. Then, cell apoptosis was analyzed by FACSCalibur flow cytometer (BD Biosciences). The results of cell apoptosis were analyzed with FlowJo v10 software. The statistical data displayed mainly included the proportions of apoptotic cells and survival cells.
Wound healing assay
The wound was scratched with a 200 μl sterile pipette tip when cells were grown to 70~80% confluence in 12-well plates. After washing with PBS, the cells were cultivated for 24 h and 48 h in the DMEM medium containing 5% FBS, respectively. The images were obtained using an inverted optical microscope (Olympus IX71, Tokyo, Japan), and the relative migration rate of cells assessed by calculating the changes of scratch width using Image-Pro Plus 6.0 software. The experiments were performed in triplicate.
Transwell assay
Transwell migration and invasion assays were assessed using 24-well transwell chambers (Millipore, Billerica, MA, USA). For transwell cell migration assay, 5×104 cells were resuspended with 200 μl serum-free DMEM medium and seeded into the upper 24-well transwell chamber. For transwell cell invasion assay, 8×104 cells were resuspended with 200 μl serum-free DMEM medium and plated on the matrigel (BD Biosciences) pre-coated 24-well transwell chambers. The lower tranwell chamber was filled with 500 ml DMEM medium containing 20% FBS. After being cultured for 24 h at 37 °C, non-migrated cells and non-invaded cells in the upper surface of the transwell chamber were slightly wiped with a cotton tip, while the migrated cells and invaded cells on the lower surface of the 24-well transwell chamber were fixed using 4% paraformaldehyde and stained with 0.5% crystal violet for 30 min. The number of migrated cells and invaded cells was photographed and counted under an inverted microscope (Olympus IX71). These experiments were performed in triplicate.
Bioinformatics analysis
The expression changes of FAM96A and FAM96B mRNA levels in various type of cancers including breast cancer was analyzed using Oncomine online database (https://www.oncomine.org/resource/login.html), which is a cancer microarray database and online date-mining platform [18]. The specific mRNA expression level of FAM96A and FAM96B between invasive breast carcinoma and normal breast tissue was analyzed in a specific dataset of breast cancer, respectively.
Statistical analysis
All the data analysis was performed using GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA). All the experiments were performed in triplicate. All the measured data were expressed as mean ± SEM. The statistically significance of the data between two independent groups was calculated using unpaired two-tailed Student′s t test. Difference with *p <0.05 was considered to be statistically significant.