2.1 Cell lines and cell culture
The human lung adenocarcinoma cell lines, A549-Mock and fucosyltranferase IV-transfected A549 (A549-Fut4) were obtained from the Department of Internal Medicine, College of Medicine, National Taiwan University. Both of these cell lines were grown in RPMI1640 (Hyclone, Thermo Scientific,) supplemented with 10%(v/v) Fetal bovine serum (Biological industries), 100 unit/mL penicillin, 0.1 mg/mL streptomycin, 0.25 μg/mL amphotericin(Biological industries), 0.15 %(w/v) sodium bicarbonate (Sigma), 10mM HEPES (Sigma), 0.25% (w/v) glucose, and 1mM sodium pyruvate(Caissonlabs, North Logan, US). They were incubated in a humidified atmosphere with 5% CO2 at 37℃. The cells were subcultured every 2-3 times per week.
2.2 siRNA knockdown
The siRNA of target genes (SLC3A2, CD166, CD44) were ordered from Thermo Scientific Dharmacon. All siRNA of these genes were dissolved in RNase-free water to produce 20 μM as stock, then stored in a -80℃ refrigerator. Before the siRNA transfection, both cell lines (A549-Mock and A549-FutIV) were seeding in 24-well plate and 5*104/mL/well. The culture medium was replaced with antibiotic-free complete medium and incubated overnight at 37℃. Then, 50μL of the siRNA in opti-medium (invitrogen) was prepared by adding 2.5μL of 5μM siRNA to 47.5μL of
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opti-medium in an 1.5mL eppendorf and gently mixed by pipetting carefully up and down, then incubated for 5 minutes at room temperature. This preparation was labeled as tube 1.
DharmaFECT transfection reagent in opti-medium was prepared by adding 1μL DharmaFECT reagent and 49μL opti-medium gently mixed and incubated for 5 minutes at room temperature, then labeled as tube 2. The content of tube 1 was then added to tube 2, mixed by pipetting carefully up and down and incubated for 20 minutes at room temperature. Then, 400 μL of antibiotic-free complete medium was added to get 500μL transfection medium with a final concentration of 25nM siRNA. This tranfection medium was prepared fresh before use. The antibiotic-free medium in the 24-well cultured plate was removed and 500 μL of fresh transfection medium added to each well.
The cell were incubated for 24 hours.
2.3 RNA preparation, reverse transcription polymerase chain reaction (RT-PCR) and
quantitative PCR(qPCR)
2.3.1 RNA preparation
The total RNA of A549-Mock and A549-Fut4 in the 24-well plate were extracted using a TRIzol Reagent (Invitrogen) following the protocol provided by the manufacturer. Briefly, the medium was removed, the cells were washed by PBS twice and then 1 mL TRIzol reagent was added to each well. The cell lysate was incubated 5
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minutes to homogenize the RNA and then transferred to a new tube. Two hundred microliters of chloroform was added into 1 mL of homogenized mixture, which was shaken vigorously, and then centrifuged at 12000g for 15 minutes at 4℃ to separate the RNA into an aqueous phase, DNA into an internal phase and protein into an organic phase. For RNA precipitation, the aqueous phase was transferred into a new tube and the tube was inverted several times after adding isopropyl alcohol. Then, the aqueous-isopropyl alcohol mixture was centrifuged at 12000g for 10 minutes at 4℃, producing an RNA pellet at the bottom of tube. The supernatant was discarded and 75%
ethyl alcohol added twice to wash the pellet. Finally, the RNA pellet was dried out and dissolved in 50 μL RNase-free water..
2.3.2 Reverse transcription polymerase chain reaction (RT-PCR)
To obtain 1μg cDNA, the total RNA concentration obtained by the previous procedures was measured using NanoDrop(Thermo scientific) and appropriate volume of total RNA was used as template in a sterile PCR tube. One hundred pmol of oligodeoxythymidine primer (oligo(dT)18) was mixed with template RNA and filled with RNase-free water to 12.5 μL. The mixture would chill on ice after incubating at 65℃ for 5 minutes. Meanwhile, pre-mix was prepared, which contained 4μL of 5X reaction buffer, 0.5μL of RNaseOUT, 2μL of 10mM dNTP mix and 1μL of RevertAid reverse transcriptase (Fermentas). These two mixtures were mixed to a total volume of
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20μL and the mixture incubated at 42℃ for 60 minutes. Finally, the reaction was
terminated by heating at 70℃ for 10 minutes and used in further experiments.
2.3.3 Quantitative PCR(qPCR)
The qPCR probe for these genes (Fut4, SLC3A2, CD166, CD44) were designed by LightCycler Probe Design Software (Roche) and the sequence of these genes were obtained from NCBI(accession number: NM_002033.3, AB018010.1, NM_001627.3, and NM_000610.3, respectively). The replicon of qPCR length was generally about 160-180 base pairs and the melting temperature was about 60℃. To avoid primer dimer, the probes used did not have ΔG less than -2000, a threshold value estimated by the software. The probes used in this study are shown in the following table.
2.4 Migration/Invasion assay
2.4.1 Wound healing assay
A549-mock and A549-FutIV cell lines were seeded at a density of 2 × 105cells primer name F primer 5'->3' R primer 5'->3'
Fut4 CCCAgACCgTgCCAACTA ggAggTgATgTggACAgC SLC3A2 CCAgAAggATgATgTCgCT CAACCTgAgTggAgAACC
ALCAM ggCAgTggAAgCgTCATA AgCAgAgACATTCAAggAgT
CD44 TCAACAgTggCAATggAgC gCAggTTCCTTgTCTCATCA
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per well in a 24-well plate and grown overnight to confluence in complete medium. The monolayer was scratched with a pipette tip and washed with PBS to remove floating cells. The scrape was monitored and photographed after 4, 8, 12, 24 h incubation.
2.4.2 Invasion assay
A cell invasion assay was performed using a Boyden chamber (Millpore, Co., USA) with 8 μm pore polycarbonate filters that were coated with 40μg geltrex (Invitrogen)
which was diluted by serum-free RPMI-1640. After 72 hrs of transfection, 2*104 cells were resuspended by 200μL serum-free medium and seeded to the upper compartment
of the Boyden chamber. The lower chamber was filled with 1 mL of RPMI-1640 complete medium. The cells were allowed to migrate for 48 hrs, incubated at 37℃, 5%
CO2. The cells were then removed from the upper chamber using a cotton swab. The cells on the lower surface of the chamber were stained with Liu’s stain and counted.
Data representing the average number of cells per pixel in five fields were compared between the siRNA groups, NC groups, and blank control groups.
2.5 Adhesion assay
Ninety-six-well plates were coated with Collagen (Cohesion; Vitrogen), Fibronectin
(Sigma-Aldrich), Gelatin (Sigma-Aldrich) and BSA for 12 hours at 4°C. Each coating protein was dissolved in PBS (pH: 7.4) to yield a final concentration of 60 μg/mL, and a volume of 100 μL was added to each individual wells. The plates were then blocked
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with 10 mg/mL Bovine serum albumin (Sigma-Aldrich) which was heated at 85℃ for 10 minutes in PBS for 1 hour at 37°C and washed with PBS twice.
Targeted knockdown cells were isolated by trypsinization and washed once in RPMI1640 with 10% FBS to stop trypsin activity. Then the cells were resolved in serum-free RPMI1640 to remove serum components. Suspensions of 104 cells/mL viable targeted knockdown cells were then added to each well and allowed to attach for 1 hour at 37°C, 5% CO2. To determine the cell adhesion ability, plates were then carefully washed three times with PBS and fixed by 5% paraformaldehyde for 15 minutes. And then they were washed by ddH2O three times and stained by crystal violet for 30 minutes. Then, they were washed by ddH2O three times and resolved crystal violet by 10 % acetic acid. Finally, the optical density was measure at 570 nm.
2.6 Protein extraction
The organic phase from the RNA extraction which contained protein was added to
300 μL of 100% ethanol per 1 mL of TRIzol reagent and then mixed by inverting the
sample. The mixture was stored for 3 minutes at room temperature and centrifuged for 2000 g for 5 minutes at 4℃. The supernatant was transferred to a new eppendorf and then 1.5 mL of isopropanol was added for homogenization. The mixture was stored at room temperature for 15 minutes and centrifuged at 12000 g for 10 minutes at 4℃. The supernatant was removed and the protein pellet was washed by a solution containing
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0.3M guanindine hydrochloride in 95% ethanol three times. During each wash step, the protein pellet was stored in wash solution for 20 minutes at room temperature and centrifuged at 7500 g for 5 minutes at 4℃. After the final washing, the protein pellet was vortexed in 2 ml absolute ethanol and allowed to sit for 20 minutes at room temperature. In the final step, it was centrifuged at 7500g for 5 minutes at 4℃ and the supernatant was discarded. The protein pellet was resolved by the solution with 9.5 M Urea and 2% CHAPS and the sample was stored at -20 ℃ for further use.
2.7 Western blot
Protein samples (15 μg) were separated by SDS-PAGE and transferred to a PVDF membrane The membrane was blocked with 5% (w/v) BSA (sigma) in PBST for 1hr at room temperature, incubated with (1) 200 ng/mL rabbit polyclonal IgG to SLC3A2 (santa cruz, CA. U.S.A.), (2) 200 ng/mL mouse monoclonal IgG to CD166 (santa cruz, CA. U.S.A.), or (3) 400 ng/mL rabbit polyclonal IgG to CD44 (santa cruz, CA. U.S.A.)
in PBST involving 5% (w/v) BSA overnight at 4 ℃. The membrane was washed with
PBST for 30 mins twice and incubated with (1) 80 ng/mL Goat polyclonal secondary antibody to rabbit IgG HRP (Abcam, Cambridge, UK), or (2) 200 ng/mL rabbit polyclonal secondary antibody to Mouse IgG HRP (Abcam, Cambridge, UK) in PBST involving 5% BSA (v/v) for 1hr. The membrane was washed with PBST for 30 minutes twice. The immunoreactive bands were visualized by film exposure (GE Healthcare,
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Uppsala, Sweden) through the ECL enhanced chemiluminescence detection system.
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