Cell culture
A549, H1650, H1299, H1975, HCC827 cell lines were purchased from Chinese Academy of Sciences Shanghai Cell Bank. PC9 was a kindly gift from Dr. Sui Yuxia (Fujian Provincial Hospital, China). All cell lines were cultured in RPMI-1640 (Hyclone) supplemented with 10% FBS (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco). Cells were incubated at 37℃ with 5% CO2.
Plasmids preparation, siRNA, shRNA and transfection
The Flag-FOXM1 lenti-virus was purchased from GENECHEM, China. Si-Snail was purchased from RaboBio, China. Dual luciferase report vector pEZX-basic and pEZX-CDH1-2000 were purchased from GeneCopoeia. Different truncations of E-cadherin promoters were amplified from pEZX-CDH1-2000 and sub-cloned to pEZX-basic by In-fusion cloning kit (Vazyme, China). The primers used for promoter cloning were as follows: Forward CDH1-1500: 5’-catttctctactagtacgcgtTCTCCTGACCTCGTGATCTGCC-3’; Forward CDH1-1000:5’-catttctctactagtacgcgtAAATTAGGCCGCTCGAGGC-3’; Forward CDH1-500: 5’-catttctctactagtacgcgtGCCCCGACTTGTCTCTCTACAA-3’; all the Reverse primer was CDH1+35:5’-catggtggcggatcctctagaCAAGCTCACAGGTGCTTTGCA-3’.
Two shRNAs that target FOXM1 were cloned to pLKO.1. After annealing the forward and reverse primers, their ends were phosphorylated with PNK (NEB). The annealed double-strand DNA were ligated to the AgeI and EcoRI double-digested pLOK.1. The forward sh1 primers was: 5'-CCGGCGCTACTTGACATTGGACCAATTCAAGAGATTGGTCCAATGTCAAGTAGCGTTTTTTGGTACC-3', reverse was: 5'-AATTGGTACCAAAAAACGCTACTTGACATTGGACCAATCTCTTGAATTGGTCCAATGTCAAGTAGCG-3'; The forward sh2 primers was: 5'-CCGGGCGTATTTCCTTAGCTCATTATTCAAGAGATAATGAGCTAAGGAAATACGCTTTTTTGGTACC-3', reverse was: 5'-AATTGGTACCAAAAAAGCGTATTTCCTTAGCTCATTATCTCTTGAATAATGAGCTAAGGAAATACGC-3'.
All of the plasmids, siRNAs and shRNAs were transfected using lipofectamine 2000 (Thermo Fisher) and performed according to the manufacturer’s instruction.
Western blot analysis
Cells lysates were prepared by RIPA (Solarbio, China) with %1 PMSF (Solarbio, China). Protein samples was separated by 10% SDS-PAGE (Beyotime Biotechnology, China) and transferred onto PVDF membranes. The membranes were blocked in 5% skim milk in TBST at room temperature for 2 h and then incubated with primary antibodies at 4 ̊C overnight. After 3 times washing with TBST, membranes were incubated with Goat anti-rabbit secondary antibodies (1:4,000 in TBST) at room temperature for 2h. After 3 times washing with TBST, the target proteins were visualized using enhanced chemiluminescence kit (Proteintech) according to the manufacturer’s protocol. Antibodies against FOXM1 and GAPDH were purchased from Proteintech. WB-used antibodies against E-cadherin, N-cadherin, Vimentin, Snail, ZEB1 were included EMT-sampler Kit, which was purchased from Cell Signaling Technology.
Immunofluorescence
Cells grown on slides were fixed in 4% paraformaldehyde for 15 min, and then washed 3 times in PBS for 5 min each. The cells were permeabilized by treatment with 0.5% triton for 10 min, and then washed 3 times with PBS for 5 min each. Cells were blocked for 30 min using undiluted goat serum. Next, cells were incubated with primary antibody (0.5 mg /mL BSA dilution, 1: 200) overnight at 4 ℃. After rinsed 3 times with PBST, the secondary antibody (0.5 mg / mL BSA dilution, 1: 200) was added and incubated at room temperature for 2h in the dark. After 3 times washed with PBST, nuclei were stained for 10 min, then rinsed with PBS 3 times. Then the slides were observed using fluorescence microscope. Antibodies used in immunofluorescence including FOXM1, Vimentin, E-cadherin, Snail and Cy3-linked anti-rabbit were purchased from Proteintech.
Migration assay
Cells were trypsinized and grown as a confluent monolayer in six-well plates. The next day, cells were scratched using a 10μL pipette tip against a ruler. Wash 3 times with PBS to remove the cells that had been scratched. RPMI-1640 complete medium, drugs or reagents were added. Took pictures under an inverted microscope at 0h and 24h and Image J was used to analyze the scratch area.
Invasion assay
The tips used must be placed at -20 ℃ for more than 2h in advance. Dilute Matrigel gel (Corning) by 1: 8 on ice, and spread 60μL of gel per chamber. Set the gel in a 37℃ incubator for 1h. The cell density was adjusted to 2 x 105 cells/mL with serum-free medium, and 100 μL was added into the invasion chamber. Add RPMI-1640 complete medium (containing 10% FBS) to the lower chamber and incubate for 24 h. Discard the small chamber culture medium and wash twice with PBS. Wipe the Matrigel and cells on the upper surface with a cotton swab, fix with 4% paraformaldehyde for 20 minutes, stain with 0.1% crystal violet for 20 minutes, and wash with water for more than 3 times. Five fields of each well were photographed under an inverted microscope and counted.
Dual luciferase report assay
Cell lysates were prepared 48h post transfected with different truncations of E-cadherin promoters in dual-Luciferase Reporter vector, and luciferase activity was measured by Dual-Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s instruction. In si-Snail experiments, the cells were transfected with siRNAs 24h prior to luciferase plasmids transfection.
Chromatin Immunoprecipitation (ChIP) Assay
ChIP was performed as described in ChIP Kit (Beyotime Biotechnology, China). In brief, 4μg FOXM1 antibody was used to immunoprecipitate DNA, then PCR was performed to amplify E-cadherin promoter. Two primers were used: Forward, 5’- CAACAGCATAGGGAGACATT-3’, Reverse, 5’ -TGTAGAGCTTCATGGGTTAGT-3’.
Proliferation assay
5000 cells were seeded into a 96-well plate with a volume of 200 μL per well. The outermost periphery of the 96-well plate was filled with 200 μL of PBS. After 24h incubation at 37 ℃, thiostrepton (Yuanye Bio, Shanghai, China) was added to the final concentrations of 0, 1, 2, 5, 10, 20, 30, 50, 75, 100μM, 6 bio-replicates for each concentration. After the end of the culture at 24h or 48h, add 20μL of MTT solution (Biosharp, 5mg/ml in PBS) to each well. Carefully discard the culture supernatant after 4 h incubation. Add 150μL DMSO to each well and shake for 10 minutes to fully melt the crystals. The wavelength of 490nm was selected to measure the results using microplate Reader (BioTec Instruments, Inc).
Colony formation assay
Cells were splited to 6 wells plates, 300 cells/well. After 24 hours of incubation, thiostrepton was added to make the final concentrations of 0, 10, 20, and 30μM, with triplicates for each concentration. After 48 h culturing in a 37 ℃, replaced all wells with RPMI-1640 complete medium. After continuous cultured for 14 days, discarded the supernatant and carefully washed twice with PBS. Add 1% crystal violet (0.5g crystal violet in 5mL 95% ethanol) and stain for 5 minutes, then slowly wash away the staining solution with tap water and dry at room temperature. Colonies were counted using Image J.
Statistical analysis.
Student’s t test was used to determine significance between two groups, whereas comparisons between multiple groups were performed using two-way ANOVA. GraphPad Prism 6 was used to perform the statistical analysis. Significance: *p≤0.05, **p≤ 0.01, ***≤ 0.001, ****≤0.0001.