Supplementary Materials and Methods
Sphere formation assay. Cellswere resuspended in serum-free medium DMEM-F12
(Invitrogen, Carlsbad, California), supplemented with 4μg/ml insulin (Sigma, St Louis, MO),
20ng/ml epidermal growth factor (Peprotech, Rocky Hill, NJ), 20 ng/ml basic FGF
(Peprotech, Rocky Hill, NJ), B27 (1:50 Invitrogen, Carlsbad, California) and 0.4 % BSA.
Cells were plated onto ultralow attachment plates (Corning, New York, USA). Every three
days, 200l 0.8% BSA F12 medium containing EGF, insulin was added. 14 days after
seeding, the number of spheroids was measured under a microscope. For serial passage of
spheres, the spheres were collected by centrifugation, dissociated into single cell with trypsin
and cultured under conditions described above.
Flow cytometry analysis. For cell surface marker analysis, cultured cells were trypsinized,
washed, and resuspended in PBS supplemented with 2% fetal bovine serum. Cells were then
incubated with anti-EpCAM antibody (Calbiochem, La Jolla, CA), PE anti-CD133
(MiltenyiBiotec, Auburn, CA), FITC anti-CD90 (Biolegend, San Diego, CA),PE
anti-CD44(eBioscience, San Diego, CA)and PE anti-CD13(Biolegend, San Diego, CA)to
detect EpCAM, CD133, CD90, CD44 or CD13 and analyzedwith the BD FACS Aria cell
sorting system (BD Biosciences, Bedford, MA). For side population (SP) analysis of cells,
cells were detached from the dishes with 0.25% trypsin, washed twice with PBS,
resuspended in DMEM medium supplemented with 10% fetal bovine serum at a
concentration of 1×106 cells/ml and culture incubated at 37℃ in a 5% CO2 incubator for 10
min. Cells were incubated at 37°C for 90 minutes with 20 μg/mL Hoechst 33342 (Sigma, St
Louis, MO), either alone or in presence of 50μmol/L Verapamil (Sigma, St Louis, MO).Cells
were washed, centrifuged and resuspended in ice-cold PBS. Then, 1μg/mL propidium iodide
(Sigma, St Louis, MO) was added and filtered through a 40μm cell strainer (BD Falcon) to
obtain single suspension cells.
Cell viability and chemo-resistance assay.Cells infected with LV-shTip30 and LV-shNon
were detached, counted and seeded into 96-well plates at 5000 cells per well. 24 hours later,
fluorouracil, pharmorubicin or cisplatin was obtained from Changhai-Hospital and added
into the medium with their corresponding IC 50 (7500M, 1.72M, and 13.33M). Medium
was removed and washed with PBS twice after the designed time point, respectively (24h,
48h, 72h, and 96h). Cell viability was determined by MTS assay reagent (CellTiter 96
AQueous one Solution Cell proliferation Assay; PromegaCorperation, USA) according to
manufacturer’s protocol. In addition, various concentrations of the three chemotherapy
agents were added, and after 72 hours cell viability was determined in the same way.
RNA extraction and reverse transcription PCR.Total RNA was isolated by NucleoSpin
RNA II kit (Macherey&Nage, Easton, PA; includes DNase I treatment). First-strand cDNA
was generated using the PrimeScript RT reagent Kit (Takara Bio, Tokyo, Japan). Analysis of
mRNA levels was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems,
Carlsbad, California) with SYBR Green-based real-time PCR. Actin was used as an
endogenous control to normalize the amount of total RNA in each sample. The primer
sequences are provided as follows (F, forward; R, reverse):
Gene symbol
TIP30
Bim1
Actin
E-cad
Nanog
Oct4
Slug F
Snail1F
Sox2
ABCG2
ABCC1
ABCB1
SIP
Twist
Zeb1
Foxc2
Primer Sequence (5'-3')
F:TCACCTTCGACGAGGAAGCT
R: GCTCTGCAGACTTCAGACCA
F: TGGAGAAGGAATGGTCCACTTC
R: GTGAGGAAACTGTGGATGAGGA
F: CGTGGACATCCGTAAAGACC
R: ACATCTGCTGGAAGGTGGAC
F: TGCCCAGAAAATGAAAAAGG
R: GTGTATGTGGCAATGCGTTC
F: GATTTGTGGGCCTGAAGAAA
R: TTGGGACTGGTGGAAGAATC
F: CTTGCTGCAGAAGTGGGTGGAGGAA
R: CTGCAGTGTGGGTTTCGGGCA
F: GGGGAGAAGCCTTTTTCTTG
R: TCCTCATGTTTGTGCAGGAG
F: AATCGGAAGCCTAACTACAGCG
R: GTCCCAGATGAGCATTGGCA
F:GCGAACCATCTCTGTGGTCT
R:GGAAAGTTGGGATCGAACAA
F:CAGGTGGAGGCAAATCTTCGT
R:ACCCTGTTAATCCGTTCGTTTT
F:CTCTATCTCTCCCGACATGACC
R:AGCAGACGATCCACAGCAAAA
F:TTGCTGCTTACATTCAGGTTTCA
R:AGCCTATCTCCTGTCGCATTA
F:CAAGAGGCGCAAACAAGCC
R:GGTTGGCAATACCGTCATCC
F:GTCCGCAGTCTTACGAGGAG
R:GCTTGAGGGTCTGAATCTTGCT
F:CTACAACAACAAGACACTGCTGT
R:TGTTCTTTCAGAGAGGTAAAGCG
F:CCTCCTGGTATCTCAACCACA
R:GAGGGTCGAGTTCTCAATCCC
Immunoprecipitation.Cell extracts were prepared by sonicating in 25 mmol/L TrisCl
(pH7.4), 100 mmol/L NaCl, 0.15% NP40, 0.25 mmol/L EDTA, and 10% glycerol,
supplemented with protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO). After
centrifugation at 14,000 rpm for 20 min, either rabbit polyclonal anti-His antibody (Cell
Signaling Technology, Danvers, MA), anti-TIP30 antibody (generated in our lab1), or IgG1
(1.5 mg antibody/mg extract, Santa Cruz Biotechnology, Santa Cruz, CA) and protein
A-agarose (Invitrogen, Carlsbad, California) were added to the supernatant cytoplasmic
lysates and incubated at 4 ℃ overnight. The beads were washed three times in the same
lysis buffer. Theproteins remaining on beads were analyzed by immunoblotting with relevant
antibodies.
Microarray analysis.Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad,
California) and purified with an RNeasy kit (Qiagen, Hilden, Germany). Microarray analysis
was performed using the NimbleGen Gene Expression Microarray, with the assistance of the
Kangchen biotechnology company. Scanning was performed with the Axon GenePix 4000B
microarray scanner. Raw data were extracted as pair files by NimbleScan software (version
2.5). Heat map of relative enrichment scores were derived by gene set enrichment analysis.
Core enrichment genes that contributed most to the enrichment result in gene set of 71
EMT-related and stemness-related genes were hierarchical clustered using Cluster 3.0
software.
Immunofluorescence staining. Cells were seeded in 24-well plates fixed with 4%
paraformaldehyde for 20-30 minutes at room temperature ,then washed with 1×PBS three
times and blocked with blocking solution (0.5% Triton X-100/1% BSA/ 1×PBS ) for 1 hour
at room temperature. For immunofluorescence staining, cells were incubated with primary
antibodies: Snail (1:200, Abcam, Cambridge, MA), E-cadherin (1:50, Cell Signaling
Technology, Danvers, MA) and Vimentin (1:100, Cell Signaling Technology, Danvers, MA)
at 4℃ overnight on shaker. The next day, after being washed three times, cells were
incubated with goat anti-mouse IgG (1:500，Invitrogen, Carlsbad, California) or goat
anti-rabbit IgG (1:500, Invitrogen, Carlsbad, California) for 1 hour, and then stained with
DAPI (Invitrogen, Carlsbad, California). All matched samples were photographed (control
and test) using immunofluorescence microscope.
Wound-healing, cell Invasion and migration assay.For wound-healing assay, the cells
were first seeded in 6-well culture plates. A wound was made in the confluent monolayer
with a plastic pipette tip and the migration of the cells at the wound front was photographed
using an inverted microscope at indicated times after the scratch.
Ability of cell invasion and migration was evaluated by the Cell Invasion Assay
Kit(Millipore) and Transwell® Permeable Support (Corning) according to themanufacturer’s
directory. Five microscopic fields were randomly selected tocalculate the total count of the
invaded or migrated cells. The relative number of cellshaving penetrated the ECM or
basement membrane was used to denote the invasion ormigration ability of the cells. All
assays were carried out three times.Two independent investigators were blinded when
reading the migration and invasion assays.
5-Aza-2’-deoxycytidine (5-Aza-2_dC) treatment. HCC cells were seeded in 6-well plate
and cultured in medium supplemented with 5-Aza-2_dC (Sigma-Aldrich, St. Louis, MO) at
the indicated concentrations for 4 days and then subjected to RNA extraction.
Chemicals and reagents
Proteasome inhibitors MG132 was purchased from Sigma and used at final
concentrations of 30uM. Cycloheximide was purchased from Sigma and used at final
concentrations of 50ug/ml. MK-2206 was obtained from Selleck Chemicals and used at final
concentrations of 0.5 uM.
1
Tong X, Li K, Luo Z, Lu B, Liu X, Wang T et al. Decreased TIP30 expression promotes tumor
metastasis in lung cancer. Am J Pathol 2009; 174: 1931-1939.