Supplementary Information
LIM and SH3 protein 1 (Lasp1) is a novel p53 transcriptional target
involved in hepatocellular carcinoma
Bei Wang, Ping Feng, Ziwei Xiao, Ee Chee Ren
Materials and methods
Cell culture conditions
All cells were maintained in DMEM supplemented with 10% fetal bovine serum in a
humidified atmosphere of 5% CO2 at 37°C. All the reagents and media used in cell
culture were purchased from Invitrogen.
Reverse transcription-PCR (RT-PCR)
Cell pellets of HCC cell lines including HA22T, Huh-1, Huh-4, Tong, PLC/PRF/5,
SNU182, SNU449, SNU475, Huh-6, Mahlavu, Sk-Hep1, SNU387, SNU398, and
SNU423 were kindly provided by the WHO Immunology Center, National University of
Singapore. HPRT and GAPDH were used as the housekeeping gene for real-time
quantitative RT-PCR and semi-quantitative RT-PCR, respectively. For one-step
quantitative RT-PCR analysis, data were presented as the fold change of Lasp1
expression in each tumor tissue relative to its corresponding non-tumor sample after
normalization with a housekeeping gene HPRT. The sequences of the primers used in
RT-PCR are listed below in Table S1.
1
Table S1. Oligonucleotide sequences used in real-time quantitative RT-PCR and
semi-quantitative RT-PCR analysis
Product
GENE
Primers
Nucleotide Sequence (5' to 3')
Lasp1
Lasp1-F
Lasp1-R
GTATCCCACGGAGAAGGTGA
TGTCTGCCACTACGCTGAAA
275bp
p53
p53-F
p53-R
CCAGGGCAGCTACGGTTTC
CTCCGTCATGTGCTGTGACTG
205bp
p21
p21-F
p21-R
GACACCACTGGAGGGTGACT
GGATTAGGGCTTCCTCTTGG
331bp
HPRT
HPRT-F
HPRT-R
GTAATGACCAGTCAACAGGGGAC
CCAGCAAGCTTGCGACCTTGACCA
177bp
GAPDH
GAPDH-F
GAPDH-R
ACCACAGTCCATGCCATCA
TCCACCACCCTGTTGCTGTA
452bp
Size
Plasmid construction for Lasp1 over-expression
The full open reading frame of Lasp1 was amplified from normal colon total RNA
(Stratagene) by two rounds of PCR. The first set of primers was for gene-specific
amplification and included 12 bases of the attB1 or attB2 site on the 5’ end of forward or
reverse primer respectively, which were 5'- AAAAAGCAGGCTCAACCATGAACCCC
AACTGC -3' (forward) and 5'- AGAAAGCTGGGTAGATGGCCTCCACGTAGTTG -3'
(reverse), with the introduced attB sequences underlined. After the second PCR using
attB adaptor-primers, which were 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3'
(attB1, forward) and 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3' (attB2,
reverse), a BP recombination reaction was then performed by using the attB-flanking
Lasp1 PCR product and the attP-containing pDONRTM221 vector to create a Lasp1-221
entry clone. Further LR recombination reactions between the attL-containing entry clone
and the attR-containing destination vectors pcDNA-DEST40 produced the final Lasp1
expression construct pDEST40-Lasp1/V5.
2
Plasmid construction for Lasp1 promoter luciferase constructs
Using the genomic DNA extracted from HepG2 cells as the template, a PCR reaction was
performed to amplify a ~1.95kb fragment containing the 5’-flanking region and portion
of exon 1 (-1872 to +74bp relative to the transcription starting site), The resulting DNA
fragment was first subcloned into the pCR-BluntII-TOPO vector and then was released
by cutting with KpnI and EcoRV. The DNA fragment was inserted into luciferase vector
pGL3-Basic (Promega) at the KpnI and SmalI sites. The constructed plasmid was named
as pGL3-Lasp1-1872.
6 forward primers plus 1 common reverse primer were designed to produce a series
of truncated deletion constructs carrying different sizes of Lasp1 promoter region. PCR
amplification using pGL3-Lasp1-1872 as template DNA was performed to generate DNA
fragment of Lasp1 promoter region with different sizes carrying KpnI and HindIII
restriction sites. The PCR product was directly cloned into pCR®4-TOPO vector. Both
pGL3-Basic vector and those positive TOPO clones were digested by KpnI and HindIII.
The ligation reaction was then performed using purified linearized vector DNA and insert
DNA. The constructs with correct sequences were named as pGL3-Lasp1-1273, pGL3Lasp1-790, pGL3-Lasp1-544, pGL3-Lasp1-255, pGL3-Lasp1-97 and pGL3-Lasp1-27.
To generate the internal deletion constructs, PCR reactions were performed using
pGL3-Lasp1-790 as a template to produce two fragments upstream and downstream of
the targeted internal deletion region respectively for each of the construct. The upstream
fragments were digested with KpnI and SphI restriction enzymes, while the downstream
fragments were cut by SphI and HindIII. The gel purified enzyme-digested upstream and
3
downstream fragments were then ligated with the KpnI- and HindIII- digested pGL3Basic vector. The isolated plasmids were first screened by enzyme digestion and further
confirmed by sequencing analysis. The positive clones with correct sequences were
named as pGL3-Lasp1Δ403-371, pGL3-Lasp1Δ83-60, pGL3-Lasp1Δ62-33 and pGL3Lasp1Δ35-12, respectively. The primers used in cloning pGL3-luciferase constructs are
listed in Table S2, with the enzyme digestion sites highlighted in bold.
Table S2. Oligonucleotide sequences used in cloning pGL3-luciferase constructs
Primers
Nucleotide sequence (5' to 3')
Lasp1 full promoter (-1872bp)
Lasp1-PR-F
Lasp1-PR-R
TAAAACTGGTTCCTCCTGGGTTTTGC
GTTCCGAGAAAAGCTGGGGCGGGGAC
truncated deletion construct
Lasp1-1273F
Lasp1-790F
Lasp1-544F
Lasp1-255F
Lasp1-97F
Lasp1-27F
Lasp1-delR
GGTACCGGATGAACACAGCACGTCCA
GGTACCGATCTGCAGTCAAATGCTC
GGTACCGCAGGGTCCAAGAGGGAGG
GGTACCCAGTCAGCCTGAGAGCGCT
GGTACCCCCCGCTGTGTTTATTAGG
GGTACCGCTGCCTGTGTAGTTGCAGC
AAGCTTACTTAGATCGCAGATCTCGAGCC
internal deletion construct
Δ403-371-Ups-F
Δ403-371-Ups-R
Δ403-371-Dns-F
Δ403-371-Dns-R
Δ83-60-Ups-F
Δ83-60-Ups-R
Δ83-60-Dns-F
Δ83-60-Dns-R
Δ62-33-Ups-F
Δ62-33-Ups-R
Δ62-33-Dns-F
Δ62-33-Dns-R
Δ35-12-Ups-F
Δ35-12-Ups-R
Δ35-12-Dns-F
Δ35-12-Dns-R
AAAGGTACCGATCTGCAGTCAAATGCTCT
AAAGCATGCACCGCATTCGGGGACTTTCT
AAAGCATGCCACCCTCGACGGGCTTGAGG
AAAAAGCTTACTTAGATCGCAGATCTCGAGCCCA
AAAGGTACCGATCTGCAGTCAAATGCTCTACCA
AAAGCATGCTAAACACAGCGGGGAGGAGG
AAAGCATGCAGGCCAGTTCCCCAGCTCCA
AAAAAGCTTACTTAGATCGCAGATCTCGAGCCCA
AAAGGTACCGATCTGCAGTCAAATGCTCTACCA
AAAGCATGCCCTCCGCCCTCCTTCCCCTA
AAAGCATGCTCGCTGCTGCCTGTGTAGTT
AAAAAGCTTACTTAGATCGCAGATCTCGAGCCCA
AAAGGTACCGATCTGCAGTCAAATGCTCTACCA
AAAGCATGCCGGCTGGAGCTGGGGAACTG
AAAGCATGCCAGCCGCGGCCGCCT
AAAAAGCTTACTTAGATCGCAGATCTCGAGCCCA
4
Site-directed mutagenesis
Briefly, pCMV-p53 plasmid was utilized as the template in the PCR reactions to generate
p53 mutants with primers carrying single nucleotide mutation. The parental strands were
digested at 37°C for 1 hour with DpnI restriction enzyme provided in the kit (Stratagene).
Subsequently, the DpnI-treated DNA was transformed into XL1 Blue Supercompetent
cells. The desired clones were verified by sequencing analysis. The primers used for PCR
are listed in Table S3, with mutated sites highlighted in red.
Table S3. Oligonucleotide sequences used in site-directed mutagenesis
Primers
Nucleotide sequence (5' to 3')
p53-K120M-F
CATTCTGGGACAGCCATGTCTGTGACTTGCACG
p53-K120M-R
CGTGCAAGTCACAGACATGGCTGTCCCAGAATG
p53-R248Q-F
GGCGGCATGAACCAGAGGCCCATCCTC
p53-R248Q-R
GAGGATGGGCCTCTGGTTCATGCCGCC
p53-R273L-F
GAACAGCTTTGAGGTGCTTGTTTGTGCCTGTCCTG
p53-R273L-R
CAGGACAGGCACAAACAAGCACCTCAAAGCTGTTC
p53-R280K-F
TGTGCCTGTCCTGGGAAAGACCGGCGCACAGAG
p53-R280K-R
CTCTGTGCGCCGGTCTTTCCCAGGACAGGCACA
Western blot
The experimental cells were washed twice with PBS and then lysed in cell lysis buffer
(1% Nonidet P-40, 150mM NaCl, 50mM Tris, pH7.8) containing protease inhibitor
mixture (Roche Applied Science) and 1mM phenylmethylsulfonyl fluoride (Sigma) on
ice for 30min. The lysates were centrifuged at 13,000 rpm for 15 min at 4°C, and the
protein concentration was measured using a Protein Assay Kit (Bio-Rad). 20μg of protein
was separated on 10%-12.5% SDS-polyacrylamide gel electrophoresis, and transferred to
5
Hybond PVDF membranes (Amersham). The membranes were extensively washed with
TBST buffer (20 mM Tris, 150 mM NaCl, 0.2% Tween-20, pH 7.6), and the
immunoreactive signals were visualized by reacting with enhanced chemiluminescence
(ECLplus) reagents followed by exposure to Hyperfilm (Amersham).
Cell proliferation and colony formation assay
HCC cell lines transiently transfected with either expression plasmid or siRNA oligos
were harvested 24 hours after transfection and replated into 96-well plate in triplicates.
The cell growth rate was measured daily by using a modified MTT assay (WST-1 reagent,
Roche) according to the manufacturer’s protocol. For anchorage-independent colony
formation assay, 0.5~1×104 HepG2 or Huh-7 cells suspended in 0.25ml of 0.35% agarDMEM /10%FBS were plated in 24-well plate in triplicates overlying a 0.7% agar
bottom layer and cultured at 37°C with 5% CO2. Two to three weeks later, the colonies
were stained with p-iodonitrotetrazolium (1mg/ml, Sigma) and photographed under a
MZFL3 stereomicroscope. Colonies > 100μm in diameter were counted and analyzed
using the Leica QWin imaging software.
Cell migration and invasion assay
The migration assay was performed in microporous membrane inserts with an 8μm pore
size, while the invasion assay utilized same inserts coated with an extra layer of
ECMatrixTM, a reconstituted basement membrane matrix of proteins derived from the
Engelbreth Holm-Swarm (EHS) mouse tumor. Briefly, 24 hours after transfection, cells
were starved in serum-free medium (DMEM/0.5%FBS) for another 24 hours. Then 0.5 –
6
1.0 × 105 of cells resuspended in 250μl DMEM/0.5%FBS were added into the migration
or invasion inserts which were loaded in wells of 24-well plate containing 500μl of
DMEM/10%FBS. After cultured for 16-18 hours, the migrated or invaded cells towards
the DMEM/10%FBS to the bottom of the insert membrane were dissociated from the
membrane by incubating with Cell Detachment Buffer and subsequently lysed and
detected by CyQuant GR® dye staining. The fluorescent intensity was measured in a
fluorescent microplate reader (Tecan) using 480/520nm filter set.
Promoter luciferase reporter assay
Hep3B (p53-/-) cells were transfected with different Lasp1 promoter luciferase reporter
constructs for luciferase assay. Briefly, exponentially growing cells were plated in 96well Flat Bottom Black Polystyrene Plates (Corning Inc.) for overnight culture before
transfection. For each well, 100ng of each luciferase reporter construct were cotransfected with either wild-type (pCMV-p53) or mutant p53 (pCMV-p53-R175H)
expression vectors together with 1ng of Renilla luciferase control vector PRL-null
(Promega). The total amount of transfected DNA in each well was kept constant by
adding empty control plasmid pcDNA3.1. Transfections were done in triplicates. Thirtysix hours after transfection, the cells were washed with PBS twice, and the reporter
Firefly luciferase activity was measured using the Dual-luciferase Reporter Assay System
kit (Promega). To correct for variations in transfection efficiency, the reporter Firefly
luciferase activity was normalized to Renilla luciferase activity (F/R ratio).
Statistical analysis
7
All functional tests were carried out in triplicates and repeated three times with similar
results. Representative data was shown as mean ± SD from triplicates. Differences of
averages and percentages between controls and Lasp1 expression vector or siRNA
transfectants were statistically analyzed using Student’s t-test. P-value less than 0.05 was
considered statistically significant.
8