Materials and Methods
Materials. Antibodies specific for EBP50 (ab3452), MMP-2
(ab37150), MMP-9 (ab38898) were purchased from Abcam (San Francisco,
CA); Caspase-3 (9662), LC-3 (4108), β-catenin (8480) and
E-cadherin(14472) were purchased from CST (Cell Signaling Technology);
β-actin mAb (AC062),HRP Goat Anti-Rabbit IgG (AS014) and HRP Goat
Anti-Mouse IgG (AS003) was purchased from ABclonal Technology; The
CIA-FLS were kindly gifted by professor Yingyuan Fu from the Department
of Immunology, Medical College of Nanchang University, Nanchang, China,
The lentiviral expression plasmid pLVX-IRES-ZsGreen1 was kindly gifted
by ZhangLin Zhang from the Department of Transfusion, First Affiliated
Hospital of Nanchang University, Nanchang, Jiangxi,
China.
Cell culture. The CIA-FLS were
maintained in Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich)
supplemented with 10% fetal bovine serum (FBS). 293T cells (ATCC,
TIB-202) were maintained in RPMI 1640 medium (GIBCO, Grand Island, NY)
supplemented with 10% FBS. The cells were cultured at 37℃ with an
atmosphere of 5% CO2 and 95%
air.
Lentivirus vector construction.
The EBP50 encoding gene was amplified from RAW264.7 cells by
reverse-transcription-polymerase chain reaction (RT-PCR). The primers
containing EcoRI and BamHI sites for PCR were as follows, forward
primer: 5’-AGAGAATTC-ATGAGCGCGGACG-3’; reverse primer:
5’-CGCGGATCC-TCAGAGGTTGCTGAAGA-3’. The thermal cycling condition for PCR
was 95℃ for 5 min, 30 cycles at 94℃ for 40 s, 54℃ for 30 s and 72℃ for
90 s, followed by a final round of 72℃ for 10 min. The PCR product was
digested by EcoRI and BamHI, and ligated with linearized
Plvx-Ires-ZsGreen1 by T4 DNA ligase to form recombinant lentiviral
expression vector
Plvx-EBP50.
Lentiviral vectors package. 293T
cells were cultured into 6-well plates at 37 °C with 5% CO2 in
humidified atmosphere. After reaching 70%~80%
confluence, 293T cells were triple transfected with the lentiviral
expression vector Plvx-EBP50 and two packaging vectors pSPAX2 and pMD2.G
as a group named LV-EBP50, and Plvx-Ires-ZsGreen1, pSPAX2 with pMD2.G as
a group named LV-Plvx by using FuGene 6 transfect regent following the
manufacturers protocol. Culture supernatants were collected every 24 h
for 3 days, filtered through a 0.45-μm poresize filter, and concentrated
two times with ultracentrifugation at 50,000× g for 120min. Each group
was diluted 1:10, 1:100, and 1:1000 times to determine virus titers. The
virus particles were resuspended in sterile PBS, and stored at –80 °C
until use.
Real-time PCR. Total cellular
RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad,
CA), following the manufacturer’s instruction, then reverse-transcribed
into complementary DNA using the Prime Script RT reagent kit (Takara,
Shiga, Japan) according to the manufacturer’s instructions. The
expression of EBP50 was determined by real-time PCR using SYBR Premix Ex
Taq (Takara, Shiga, Japan) and Step One Plus Real-Time PCR System
(Applied Biosystems, CA). Primers used for PCR amplification were:
GAPDH: 5-GCACCGTCAAGGCTGAGAAC-3′ (forward), 5′-TGGTGAAGACGCCAGTGGA-3′
(reverse); EBP50: 5’-AGGCCAGTTCATCCGAGCAG-3’(forward),
5’-CAGCAGCTTGGCCTCATCAC-3’ (reverse). The mRNA levels of EBP50 relative
to the control was calculated by
2−△△CT.
Western blotting. The cellular
protein was collected and lysed using RIPA lysis buffer containing 1 mM
phen-ylmethylsulfonyl fluoride and 1% (vol/vol) protease inhibitor
cocktail (Beyotime, Haimen, Jiangsu, China).Equal amounts of proteins
were separated on sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene
fluoride membranes at 4℃for 1 h. Membranes were blocked in 5% BSA and
incubated with primary antibodies against EBP50, LC3, Caspase-3,
β-catenin, E-cadherin, Akt, MMP-2 and MMP-9 at 4℃ overnight. Then
membranes were incubated at room temperature for 1 h with relevant
secondary antibodies, and blots were visualized using enhanced
chemiluminescence (ECL; Thermo Pierce, Rockford, Illinois, USA)
according to the manufacturer’s instructions and quantified by
densitometry using Quantity One image software with β-actin used as an
internal
control.
MTT cytotoxicity assay. FLS were cultured in DMEM with 10%
heat-inactivated FBS and antibiotics at 37 °C in a humidifified
atmosphere with 5% CO2. 5×103 cells/well were plated
in a 96-well plate. After 12 h, 20 μL MTT solution (0.5 mg/mL) was added
in FLS culture mediumand incubated for 4 h. The culture mediumand in
each well was discarded and added 150 μL DMSO. The absorption of each
well was measured at 490 nm.
Phagocytosis assay by flow
cytometry (FCM). FLS transduced with or without recombinant lentivirus
for 48 h,72 h,96 h. Then cells were washed thoroughly with D-Hanks
buffer to remove extracellular recombinant lentivirus and depleted
media, at the same time, fls were transfered seedind in 96-well plates
5×103/cells with annexin V binding buffer resuspended.
Then were incubated at 2-8℃ for 15 min with 5μl Annexin V-FITC, and were
incubated at 2-8℃ for 5 min with 5μl PI, finally cells were collected
and analyzed using Cytomics FC500 flow cytometer (Beckman Coulter Inc.,
Brea, CA, USA).
In Vitro Migration and Invasion Assay of FLS . In vitro cell
migration assays were performed as described previously using Trans-well
chambers (8 μM pore size,Corning, NY, USA). When cells reached
subconfluency (75%–80%), cells were serum-starved for 24 h. After
detachment with trypsin, cells were washed with PBS and resuspended in
serum-free medium. Next, 100 μL of cell suspension
(2×105 cells/mL) was added to the upper chamber while
complete medium was added to the bottom wells. 24 h later, cells that
had not migrated were removed from the upper surface of the filters
using cotton swabs. Cells that had migrated were fixed with 5%
glutaraldehyde solution to determine the number of migratory cells. The
lower surface of the filters was stained with crystal violet. Images of
three fields were captured from each membrane, and the number of
migratory cells were counted. The mean of triplicate assays for each
experimental condition was used.
Statistical analysis. All the presented data and results were
confirmed in at least three independent experiments. Unpaired Student’s
t test or one-way analysis of variance was used to determine the
significance of theresults. Data were considered statistically
significant at P< 0.05.