Download Supporting Information - PNAS

Copy and paste this link to your website, so they can see this document directly without any plugins.



Keywords

were, cells, with, using, TIG-7, from, medium, H1299-GFP, coculture, conditioned, After, Transwell, after, performed, p53-depleted, containing, noncontact, cell, seeded, system, antibody, plated, according, 103), measured, H1299-LUC, plate, into, culture, activity

Transcript

Supporting Information
Otomo et al. 10.1073/pnas.1412062112
SI Materials and Methods
Cell Culture. Human lung cancer cell lines (H1299, H460, and
A549) were maintained in RPMI medium 1640 (Sigma-Aldrich)
supplemented with 10% (vol/vol) FBS (Gibco), penicillin (100
units/mL) (Sigma-Aldrich), and streptomycin (100 units/mL)
(Sigma-Aldrich) at 37 °C in a 5% (vol/vol) CO2 incubator. Human
embryonic lung fibroblast cell lines (TIG-7 and WI-38) were maintained in DMEM (Sigma-Aldrich) supplemented with 10% (vol/vol)
FBS, penicillin (100 units/mL), and streptomycin (100 units/mL) at
37 °C in a 5% (vol/vol) CO2 incubator. Immortalized small airway
epithelial cells (SAECs) were generated and maintained as described previously (1).
Viral Infection. Retroviral production and infection were performed as described previously (1). Briefly, the day before
transfection, Phoenix-Ampho cells (5 × 106) were seeded in 100-mm
tissue culture dishes. The next day, retroviral vectors were transfected
using Lipofectamine 2000 reagent (Invitrogen), according to the
manufacturer’s protocol. Culture media were replaced 6 h after
transfection, incubated at 37 °C for 48 h, and retrovirus-containing
supernatants were collected from culture media. TIG-7 cells were
plated in tissue culture dishes and infected with retroviruses in media
containing 4 μg/mL Polybrene (Sigma-Aldrich). After being infected,
media containing retroviruses were replaced with fresh media and,
2 d after infection, culture media were changed to media containing
0.5 μg/mL puromycin (Sigma-Aldrich).
Lentiviral production and infection were performed as described previously (1). Briefly, the ViraPower Lentiviral Expression System (Invitrogen) was used to produce lentiviruses
according to the manufacturer’s protocol, and infected cells were
selected using 4 μg/mL (for TIG-7 and WI-38) or 10 μg/mL (for
H1299) Blasticidin S (Invitrogen).
Plasmid Construction. The construction of pSR-53 and pSL-p53
was described previously (2). To construct pLenti-shTSPAN12
(pSL-TSPAN12), the following oligonucleotides containing target sequences were annealed and ligated into the pSUPER.retro
vector (Oligoengine): shTSPAN12, 5′-GATCCCCGCTTATCTTTGCCTTCTCCTTCAAGAGAGGAGAAGGCAAAGA-
TAAGCTTTTTGGAAA-3′ and 5′-AGCTTTTCCAAAAAG-
CTTATCTTTGCCTTCTCCTCTCTTGAAGGAGAAGGCAA-
AGATAAGCGGG-3′. Target sequences containing the H1
promoter were digested from the above plasmid (pSR-TSPAN12)
and ligated into the pLenti6/V5-DEST vector (Invitrogen). To
construct pLenti-TSPAN12-FHH, cDNA fragments encoding
TSPAN12 were amplified by PCR using the following primers:
5′-CCGGATCCATGGCCAGAGAAGATTCCGTG-3′ and 5′-CC-
GCTCGAGTAACTCCTCCATCTCAAAGTGT-3′. The PCR
fragment was digested with BamHI and XhoI and inserted into
the pcDNA3.1-FHH (FLAG-HA-His) plasmid. The TSPAN12
fragment containing the FHH tag was digested from the above
plasmid (pcDNA3.1-TSPAN12-FHH) and ligated into the
pLenti6/V5-DEST vector (Invitrogen). To construct pFUSETSPAN12-LEL-Fc and pFUSE-TSPAN12-LEL-VR-Fc, cDNA
fragments encoding TSPAN12-LEL and TSPAN12-LEL-VR
were amplified by PCR using the following primers: TSPAN12LEL, 5′-GGCCATGGTTGGCGTTTGGACATATGAACAGGA-
AC-3′ and 5′-GCCGGATCCGCTCTGAAAGTACAGATCCTC-
CCTCAGCACCTGCAGTTGTTTGG-3′; TSPAN12-LEL-VR,
5′-GGCCATGGTTTGCTGTGGAGTAGTATATTTCACTG-3′ and
5′-GCCGGATCCGCTCTGAAAGTACAGATCCTCACAACCC-
TCTTGATAAAGGTCAC-3′. PCR fragments were digested with
NcoI and BamHI and inserted into the pFUSE-hIgG4-Fc2 vector (InvivoGen) digested with NcoI and BglII.
Antibodies. Anti-p53 antibody (DO-1)-conjugated horseradish
peroxidase and an antiactin antibody were purchased from Santa
Cruz Biotechnology. The anti-p21waf1 antibody was from Epitomics. The anti–β-catenin antibody was from Cell Signaling
Technology. The anti–α-tubulin and anti–α-smooth muscle actin
antibodies were from Sigma-Aldrich. The anti-CXCL6 antibody
and mouseIgG1 isotype control were from R&D Systems. Another anti-CXCL6 antibody was from Novus Biologicals. An
anti-TSPAN12 antibody was generated by immunizing with
rabbits the C-terminal peptides of TSPAN12 (EHTSMANSFNTHFEMEEL) and immunized antiserum was affinity purified.
siRNA Transfection. siRNA duplexes were purchased from SigmaAldrich (for TSPAN12, β-catenin, and CXCL6) and Invitrogen
(for p53). The sequences of siRNA duplexes were as follows:
si-TSPAN12#1, 5′-GCUUAUCUUUGCCUUCUCCTT-3′ and
5′-GGAGAAGGCAAAGAUAAGCTT-3′; si-TSPAN12#2,
5′-AUGAGGGACUACCUAAAUATT-3′ and 5′-UAUUUA-
GGUAGUCCCUCAUTT-3′; si-β-catenin#1, 5′-GGAUGUUCACAACCGAAUUTT-3′ and 5′-AAUUCGGUUGUGAA-
CAUCCTT-3′; si-β-catenin#2, 5′-CCACUAAUGUCCAGCGUUUTT-3′ and 5′-AAACGCUGGACAUUAGUGGTT-3′;
si-CXCL6#1, 5′-GGAGGUAUCCUGUUGUUCUTT-3′ and
5′-AGAACAACAGGAUACCUCCTT-3′; si-CXCL6#2, 5′-CG-
CGUUACGCUGAGAGUAATT-3′ and 5′-UUACUCUCAG-
CGUAACGCGTT-3′; si-p53#1, 5′-UUAACCCUCACAAUG-
CACUCUGUGA-3′ and 5′-UCACAGAGUGCAUUGUGA-
GGGUUAA-3′; si-p53#2, 5′-CCAUCCACUACAACUAC-
AUGUGUAA-3′ and 5′-UUACACAUGUAGUUGUAGU-
GGAUGG-3′. The transfection of siRNAs in fibroblasts was
performed using Lipofectamine 2000 (Invitrogen) or Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s
protocol.
Immunoblot Analysis. To produce conditioned medium, lung
cancer cells (2 × 106) were seeded on 100-mm dishes and conditioned medium was collected after 48 h. Fibroblasts (2 × 105)
were seeded on six-well plates and treated with conditioned
medium or transfected with siRNAs. After 24–72 h, cells were
harvested, washed with PBS, and lysed with lysis buffer [50 mM
Tris·HCl (pH 7.2), 250 mM NaCl, 2 mM MgCl2, 0.1 mM EDTA,
0.1 mM EGTA, and 0.1% Nonidet P-40] containing proteinase
inhibitor mixture (Roche), 10 mM NaF, 1 mM Na3VO4, and
1 mM DTT. After being incubation for 30 min on ice, lysates
were centrifuged at 20,000 × g for 15 min and the supernatants
collected were mixed with SDS/PAGE sample buffer. The samples were then boiled for 5 min and resolved by 5–20% (wt/vol)
SDS/PAGE. After electrophoresis, the proteins were transferred
to a PVDF membrane (Millipore), blocked with 5% (wt/vol)
nonfat milk in TBST [20 mM Tris·HCl (pH 7.6), 137 mM NaCl,
0.1% Tween-20], and probed with primary antibodies at room
temperature for 1 h. The blots were then washed, exposed to
HRP-conjugated secondary antibodies (1:5,000 dilution) for 1 h,
and the antigen–antibody complex was detected by enhanced
chemiluminescence (Amersham Pharmacia Biotech).
Quantitative RT-PCR. Total RNA was isolated from cells using the
RNeasy mini kit (Qiagen). Two micrograms of total RNA was
reverse transcribed with the SuperScript III First-Strand Synthesis
system (Invitrogen). Quantitative RT-PCR (qRT-PCR) was
Otomo et al. www.pnas.org/cgi/content/short/1412062112 1 of 10
performed using the FastStart Universal SYBRGreenmaster mix
(Roche) and analyzed with the 7900HT Fast Real-Time PCR
system (Applied Biosystems) or CFX96 Touch Real-Time PCR
Detection system (Bio-Rad). The primers for qRT-PCR were as
follows: 5′-GTGAAGGTCGGAGTCAACG-3′ and 5′-TGAGGTCAATGAAGGGGTC-3′ for GAPDH; 5′-TAGTGTGGTG-
GTGCCCTATGAG-3′ and 5′-AGTGTGATGATGGTGAGGA-
TGG-3′ for p53; 5′-TGATTAGCAGCGGAACAAGG-3′ and
5′-CGTTAGTGCCAGGAAAGACAAC-3′ for p21waf1; 5′-TGGGAGTAGGATGTGGTGAAAG-3′ and 5′-AAGAGCAGATT-
GAGGGCGTAG-3′ for TSPAN12; 5′-CCCACTGGCCTCTGA-
TAAAGG-3′ and 5′-ACGCAAAGGTGCATGATTTG-3′ for
β-catenin; and 5′-ATTTCCCCAGCATCCCAAAG-3′ and
5′-CATAGTGGTCAAGAGAGGGTTCG-3′ for CXCL6.
Semiquantitative RT-PCR. Total RNA was isolated from cells using
the RNeasy mini kit (Qiagen). Two micrograms of total RNA was
reverse transcribed with the SuperScript III First-Strand Synthesis
system (Invitrogen). The PCR was performed using Taq DNA
Polymerase (1 unit/μL), dNTPPack (Roche) in a PCR thermal
cycler (MJ Research, Bio-Rad). The primers for the PCR were
as follows: TSPAN12, 5′-ATGGCCAGAGAAGATTCCGTG-3′
and 5′-TTATAACTCCTCCATCTCAAAGT-3′; p53, 5′-AAA-
GGGGAGCCTCACCACG-3′ and 5′-ACGCACACCTATTG-
CAAGCAA-3′; p21waf1, 5′-AGTGGACAGCGAGCAGCTGA-
GC-3′ and 5′-GCAGCAGAGCAGGTGAGGTGC-3′; CXCL6,
5′-ATGAGCCTCCCGTCCAGCC-3′ and 5′-TCAGTTTTTCT-
TGTTTCCACTGT-3′; HGF, 5′-GGTTCTCAATGTTTCCCA-
GCTG-3′ and 5′-CTATGACTGTGGTACCTTATATG-3′; TFPI2,
5′-ATGGACCCCGCTCGCCCC-3′ and 5′-TTAAAATTGCTTC-
TTCCGAATTTTC-3′; DLL4, 5′-CTGCTGGTACTGCTGGG-
CA-3′ and 5′-TTATACCTCCGTGGCAATGAC-3′; TNFRSF19,
5′-GGTTGTGGGGTGCATTCTGC-3′ and 5′-TCACAGGGA-
ACCCAGTCGCT-3′; SALL1, 5′-ACTGCTTGTGACATTTGT-
GGC-3′ and 5′-TTAACTCGTGACGATCTCCTTG-3′; PTGS1,
5′-GAACATGGACCACCACATCCT-3′ and 5′-TCAGAGC-
TCTGTGGATGGTC-3′; NCKAP5, 5′-TCCAGTCAG-CCC-
TTTCTGCA-3′ and 5′-TCAAGTTGTCTCAATTTCTGGG-3′;
and GAPDH, 5′-GGGGAGCCAAAAGGGTCATCATCTC-3′
and 5′-TCCACAGTCTTCTGGGTGGCAGTGA-3′. The PCR
products were subjected to electrophoresis on a 1% agarose gel
containing 0.5 μg/mL ethidium bromide.
ELISA Assay. TIG-7 cells (5 × 104) were seeded on 24-well plates
and transfected with siRNAs. Culture medium was replaced 48 h
after transfection, further incubated for 48 h, and conditioned
medium was then collected. The antigen–antibody reaction was
performed using DuoSet ELISA for human CXCL6 (R&D Systems) according to the manufacturer’s instructions. Streptavidin–
HRP-bound samples were reacted with peroxidase (Sumilon),
and measured at an absorbance of 490 nm using a plate reader
(PerkinElmer).
Invasion Assay by Coculture in Matrigel. In the contact coculture,
H1299-GFP cells (1 × 103) and TIG-7 cells (1 × 103) were suspended in 50 μL of a mixture of DMEM and Matrigel (BectonDickinson) (1:1, vol/vol) and layered onto 20 μL of presolidified
mixture in a 96-well Transwell plate (Corning). In the noncontact coculture, TIG-7 cells (3 × 103) were seeded on the
bottom plate and H1299-GFP cells (1 × 103) were layered onto
a 96-well Transwell insert as described above. Culture medium
(150 μL) was added to the lower wells and changed every 2–3 d.
The anti-CXCL6 antibody (10 μg/mL) or TSPAN12-large extracellular loop (LEL)-Fc protein (10 μg/mL) was added to
culture medium and changed every 2 d. Colonies of H1299-GFP
cells were observed under a confocal microscope for 4–5 d after
culturing and colonies morphologically exhibiting the invasive
outgrowth phenotype were counted.
Proliferation Assay by Coculture. In the contact coculture, H1299LUC cells (3 × 102) were plated onto a monolayer of TIG-7 cells
(1 × 103) in a 96-well plate. The next day, 150 μg/mL D-luciferin
(Wako) was added to the culture medium and luminescence was
measured using a luminometer (PerkinElmer) to monitor the
proliferation of H1299-LUC cells. After measuring luminescence, the medium was replaced and, after 24 h, luminescence
was measured again. These procedures were repeated five times.
In the noncontact coculture using conditioned medium, H1299LUC cells (3 × 102) and TIG-7 cells (1 × 103) were seeded individually onto a 96-well plate. The next day, medium in H1299LUC cells was changed to conditioned medium collected from
TIG-7 cells. Luciferase activity was measured after 24 h, and
medium in H1299-LUC cells was changed to newly conditioned
medium from TIG-7 every day for 5 d. In the noncontact coculture using a Transwell insert, H1299-LUC cells (5 × 102) were
seeded on a 96-well companion plate and TIG-7 cells (1 × 103)
were seeded on a 96-well Transwell insert. After 24 h, the
Transwell insert was combined with the companion plate and
luciferase activity in H1299-LUC cells 24 h after being incubated
was measured using a luminometer. Medium from the companion plate was replaced and luciferase activity in H1299-LUC
cells was measured at 24-h intervals.
Transwell Migration Assay in Coculture. In the contact coculture,
H1299-GFP cells (5 × 104) were plated with TIG-7 cells (5 ×
104) suspended in 500 μL of serum-free DMEM, and then
plated onto 8.0-μm pore size 24-well Transwell inserts (BectonDickinson). The bottom well was filled with 750 μL of DMEM
containing 10% (vol/vol) FBS. After being incubated at 30 °C
with 5% (vol/vol) CO2 for 16 h, the cells on the upper surface of
the Transwell insert were scraped using cotton swabs. The
migrated cells on the lower surface were observed under
a fluorescence microscope.
Microarray Analysis. Total RNA was purified using an RNeasy
kit (Qiagen) and subjected to a microarray experiment. Target
cRNA was prepared from 5 μg total RNA with a One-Cycle
cDNA Synthesis kit and 3′-amplification reagents for IVT Labeling (Affimetrix), and hybridized to GeneChip Human Genome
U133 Plus 2.0 arrays (Affimetrix) according to the manufacturer’s
instructions. The expression value (signal) and change value (signal
log ratio) of each gene were calculated and normalized using
GeneChip Operating Software version 1.4 (Affimetrix). We performed the experiment in duplicate and selected the genes exhibiting threefold or more changes in both comparisons.
Production and Purification of Large Extracellular Loop of TSPAN12.
The 293FT cells were plated at 80–90% confluence the day before transfection and subsequently transfected with pFUSETSPAN12-LEL or pFUSE-TSPAN12-LEL-VR or no insert
control (pFUSE-Fc) using Lipofectamine 2000 according to the
manufacturer’s protocol. Conditioned meda were harvested 24–
96 h after transfection and debris were removed by centrifugation
at 600 × g for 3 min. Soluble proteins were purified with Protein A
Sepharose (GE Healthcare) and concentrated using Amicon Ultra
3K (Millipore). To more efficiently collect TSPAN12-LEL, cells
were harvested and lysed with lysis buffer (20 mM Hepes-KOH,
pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% CHAPS) 72 h after
transfection and subsequently sonicated. TSPAN12-LEL was purified with Protein A Sepharose and concentrated using AmiconUltra
3K. The concentration of purified proteins was determined with
SDS/PAGE and Coomassie Brilliant Blue (CBB) staining.
Tumor Growth Assay.H1299-LUC cells (5 × 105) were mixed with
TIG-7 cells (1.5 × 106) in 100 μL DMEM–Matrigel mixture (1:1,
vol/vol) and injected s.c. into the backs of BALB/c-nu/nu mice
(CREA Japan). Tumor growth was monitored using the IVIS
Otomo et al. www.pnas.org/cgi/content/short/1412062112 2 of 10
imaging system (Xenogen). The mouse xenograft study was conducted according to the regulations of the Institutional Animal Care
and Use Committee at the National Cancer Center Research Institute, Tokyo Japan.
Statistical Analysis.Data are shown as the means ± SDs of three or
more independent experiments. Statistical analyses were performed
using the Student t test, paired t test, and Wilcoxon signed-rank test
with a significance level of P < 0.05.
1. Otsubo C, et al. (2014) TSPAN2 is involved in cell invasion and motility during lung
cancer progression. Cell Reports 7(2):527–538.
2. Endo Y, et al. (2008) Regulation of clathrin-mediated endocytosis by p53. Genes Cells
13(4):375–386.
Fig. S1. Expression levels of p53 (TP53) in breast cancer-associated stromal tissues obtained by laser-captured microdissection were lower than those in stromal
tissues. (A and B) Expression levels of p53 in 6 samples of normal stromal tissues and 53 samples of cancer-associated stromal tissues extracted from the
Oncomine dataset are shown individually (A) and collectively (B). (C) p53 expression in TIG-7 cells was down-regulated by conditioned medium from cancer
cells. TIG-7 cells were treated with conditioned medium for 48 h, and expression levels of the indicated proteins in these cell lysates were determined by
immunoblotting and quantified using ImageJ version 1.47c software.
Otomo et al. www.pnas.org/cgi/content/short/1412062112 3 of 10
Fig. S2. p53-depleted fibroblasts enhanced the invasion and migration of cancer cells through direct cell-to-cell contact. (A) Scheme of the noncontact coculture system using a three-dimentional invasion assay. (B and C) p53-depleted TIG-7 cells did not enhance invasiveness in H1299-GFP cells regardless of cell
number and confluency using a contact coculture system. Three times as many H1299-GFP cells as in the assays of Fig. 2 D and E were cocultured with parental
TIG-7 cells or p53-depleted TIG-7 cells, or cultured alone in Matrigel. After 4–5 d, H1299-GFP cells were observed under a confocal microscope (B). (Scale bar,
100 μm.) Quantification of invasive phenotypic cells in H1299-GFP cells (C). (D) Scheme of the contact coculture migration assay. (E and F) p53-depleted TIG-7
cells enhanced migration activity in H1299-GFP cells using the contact coculture system. H1299-GFP cells were cocultured with parental TIG-7 cells or p53depleted TIG-7 cells, or cultured alone in an 8.0-μm pore Transwell insert. After 16 h, migrated cells were observed under a fluorescence microscope (E).
Quantification of migrated H1299-GFP cells (F). (G) Scheme of the noncontact coculture migration assay. (H and I) p53-depleted TIG-7 cells did not enhance
migration activity in H1299-GFP cells using the noncontact coculture system. H1299-GFP cells were plated on an 8.0-μm pore Transwell insert, and parental
TIG-7 cells or p53-depleted TIG-7 cells were plated on the bottom well. After 16 h, migrated cells were observed under a fluorescence microscope (H).
Quantification of migrated H1299-GFP cells (I). Data are the mean ± SD of three or more independent experiments. Statistical analyses were performed using
the Student t test. **P < 0.01, ***P < 0.001.
Otomo et al. www.pnas.org/cgi/content/short/1412062112 4 of 10
Fig. S3. p53-depleted TIG-7 cells did not enhance cell proliferation in H1299-LUC cells using the noncontact coculture system. (A) Scheme of the noncontact
coculture system using conditioned medium for the cell proliferation assay. (B) The cell proliferation assay using the noncontact coculture system is shown.
H1299-LUC cells were treated with conditioned medium from parental TIG-7 cells or p53-depleted TIG-7 cells. Luciferase activity was measured every day until
day 6.
Fig. S4. TSPAN12 was derepressed by p53 knockdown. (A) Expression levels altered by p53 knockdown are listed. Microarray experiments were performed
using cDNA samples from parental TIG-7 cells or p53-depleted TIG-7 cells. Fifty-one genes were up-regulated (fold change, >3) and 9 genes were downregulated (fold change, <0.33) in p53-depleted TIG-7 cells. (B) Scheme of the identification of TSPAN12. (C) TSPAN12 was derepressed by transient p53
knockdown using si-p53. Cell lysates were prepared from fibroblasts transfected with indicated siRNAs and the expression levels of indicated genes were
determined by immunoblotting. (D and E) The expression level of TSPAN12 was higher in breast cancer-associated stromal tissues than in normal stromal
tissues. (D and E) The expression levels of TSPAN12 in 6 samples of normal stromal tissues and 53 samples of cancer-associated stromal tissues extracted from
the Oncomine dataset are shown individually (D) and collectively (E).
Otomo et al. www.pnas.org/cgi/content/short/1412062112 5 of 10
Fig. S5. TSPAN12 regulated the migration activity of cancer cells enhanced by p53-depleted fibroblasts. (A and B) Efficiency of TSPAN12 knockdown in TIG-7
cells. TIG-7 cells were infected with either control lentiviruses or lentiviruses for the expression of shRNAs against p53 and transfected with control siRNAs or
siRNAs against TSPAN12. Expression levels of the indicated genes were determined by qRT-PCR (A) and immunoblotting (B). (C) TSPAN12 knockdown in p53depleted TIG-7 cells inhibited the migration activity of H1299-GFP cells. H1299-GFP cells were cocultured with either p53-depleted TIG-7 or TIG-7 cells depleted
of both p53 and TSPAN12 in an 8.0-μm pore cell culture insert. After 16 h, migrated cells were observed and quantified. Data are the mean ± SD of three or
more independent experiments. Statistical analyses were performed using the Student t test. *P < 0.05, **P < 0.01.
Otomo et al. www.pnas.org/cgi/content/short/1412062112 6 of 10
Fig. S6. TSPAN12 regulated cancer cell invasiveness and proliferation. (A) Efficiency of TSPAN12 knockdown in parental TIG-7 cells. Parental TIG-7 cells were
transfected with control siRNAs or siRNAs against TSPAN12. Expression levels of TSPAN12 were determined by qRT-PCR. (B) TSPAN12 knockdown in parental
TIG-7 cells did not inhibit invasiveness in H1299-GFP cells. H1299-GFP cells were cocultured with either parental TIG-7 cells or TSPAN12-depleted TIG-7 cells in
Matrigel. After 4–5 d, invaded H1299-GFP cells were observed and quantified. (C) TSPAN12 knockdown in parental TIG-7 cells did not inhibit proliferation in
H1299-LUC cells. H1299-LUC cells were cocultured with either parental TIG-7 cells or TSPAN12-depleted TIG-7 cells in 96-well plates. Luciferase activity was
measured every day until day 6. (D) Expression levels of TSPAN12 in TIG-7 cells infected with lentiviruses with or without TSPAN12-FHH. (E) The overexpression
of TSPAN12 in TIG-7 cells enhanced invasiveness in H1299-GFP cells. H1299-GFP cells were cocultured with either parental TIG-7 cells or TSPAN12-expressing
TIG-7 cells in Matrigel. After 4–5 d, invaded H1299-GFP cells were observed and quantified. (F) The overexpression of TSPAN12 in TIG-7 cells enhanced proliferation in H1299-LUC cells. H1299-LUC cells were cocultured with either parental TIG-7 cells or TSPAN12-expressing TIG-7 cells in 96-well plates. Luciferase
activity was measured every day until day 6. (G) Construction of large extracellular loops of TSPAN12. (H) Purified large extracellular loops of TSPAN12 were
stained with CBB. (I) The large extracellular loops of TSPAN12 inhibited the invasiveness of H1299-GFP cells. H1299-GFP cells were cocultured with either
parental TIG-7 cells or p53-depleted TIG-7 cells with indicated LELs of TSPAN12 in Matrigel. After 4–5 d, invaded H1299-GFP cells were observed and quantified.
Data are the mean ± SD of three or more independent experiments. Statistical analyses were performed using the Student t test. *P < 0.05, ***P < 0.001.
Otomo et al. www.pnas.org/cgi/content/short/1412062112 7 of 10
Fig. S7. TSPAN12 regulated tumor growth enhanced by p53-depleted fibroblasts. (A) Four weeks after the inoculation, the tumor volume (Left) and tumor
weight (Right) of the H1299-LUC xenograft tumor mixed with either parental TIG-7 cells or p53-depleted TIG-7 cells were measured (n = 4 per group, paired
t test for tumor volume *P < 0.05; n = 8 per group, Wilcoxon signed-rank test for tumor weight *P < 0.05). (Left back) Coinjection with parental TIG-7 cells.
(Right back) Coinjection with p53-depleted TIG-7 cells. (B) Four weeks after the inoculation, the tumor volume (Left) and tumor weight (Right) of the H1299LUC xenograft tumor mixed with either p53-depleted TIG-7 cells or p53-depleted TIG-7 cells expressing sh-TSPAN12 were measured (n = 5 per group, paired
t test for tumor volume *P < 0.05; n = 9 per group, Wilcoxon signed-rank test for tumor weight *P < 0.05).
Otomo et al. www.pnas.org/cgi/content/short/1412062112 8 of 10
Fig. S8. (A and B) Decrease in β-catenin in TIG-7 cells by siRNAs. p53-depleted TIG-7 cells were transfected with control siRNAs or si–β-catenin. Expression levels
of the indicated genes were determined by qRT-PCR (A) and immunoblotting (B). (C) Identification of CXCL6 as a p53-up-regulated TSPAN12-regulated gene.
Expression levels of selected genes, which have the potential to function in cancer progression, including cell proliferation, invasion, and metastasis, were
indicated using RNAs from TIG-7 cells by semiquantitative RT-PCR. Lane 1, parental TIG-7 cells; lane 2, p53-depleted TIG-7 cells transfected with control siRNAs;
lane 3, p53-depleted TIG-7 cells transfected with si-TSPAN12#1; lane 4, p53-depleted TIG-7 cells transfected with si-TSPAN12#2. (D and E) Knockdown efficiency
of CXCL6 in p53-depleted TIG-7 cells. p53-depleted TIG-7 cells were transfected with control siRNAs or si-CXCL6. The expression level of CXCL6 in cells depleted
of CXCL6 was determined by qRT-PCR (D). The production of CXCL6 secreted from cells depleted of CXCL6 was quantified by ELISA (E). (F and G) TSPAN12 and
CXCL6 were down-regulated by the treatment with nutlin-3. TIG-7 cells were treated with 10 μM of nutlin-3 for 24 h and the expression of the indicated
proteins was determined by immunoblotting (F) and ELISA (G). (H and I) The overexpression of TSPAN12 in TIG-7 cells induced a slight increase in CXCL6.
Expression levels of CXCL6 in parental or TSPAN12-expressed TIG-7 cells were determined by qRT-PCR (H) and ELISA (I). Data are the mean ± SD of three or
more independent experiments. Statistical analyses were performed using the Student t test. *P < 0.05, **P < 0.01, ***P < 0.001.
Otomo et al. www.pnas.org/cgi/content/short/1412062112 9 of 10
Fig. S9. Expression levels of β-catenin and CXCL6 were higher in breast cancer-associated stromal tissues than in normal stromal tissues. (A and B) Expression
levels of β-catenin in 6 samples of normal stromal tissues and 53 samples of cancer-associated stromal tissues extracted from the Oncomine dataset are shown
individually (A) and collectively (B). (C and D) Expression levels of CXCL6 in 6 samples of normal stromal tissues and 53 samples of cancer-associated stromal
tissues extracted from the Oncomine dataset are shown individually (C) and collectively (D).
Table S1. p53-up-regulated genes encoding membrane proteins in fibroblasts
Representative public ID Gene symbol Gene title Fold change
NM_001657 AREG / AREGB Amphiregulin / amphiregulin B 24.217
NM_007287/AI433463 MME Membrane metallo-endopeptidase 9.721/4.613*
NM_018057 SLC6A15 Solute carrier family 6 (neutral amino acid transporter), member 15 7.620
AA502609 TRPA1 Transient receptor potential cation channel, subfamily A, member 1 5.209
AB036931 DLL4 Delta-like 4 (Drosophila) 5.161
BF059512 DNER Delta/notch-like EGF repeat containing 5.160
H20055 GRIA4 Glutamate receptor, ionotropic, AMPA 4 4.596
NM_012338 TSPAN12 Tetraspanin 12 4.580
NM_000640 IL13RA2 Interleukin 13 receptor, alpha 2 4.551
X68742 ITGA1 Integrin, alpha 1 3.968
AI680986/R70320 SLITRK6 SLIT and NTRK-like family, member 6 3.618/3.541*
AI873273 SLC16A6 Solute carrier family 16, member 6 (monocarboxylic acid transporter 7) 3.612
NM_001993 F3 Coagulation factor III (thromboplastin, tissue factor) 3.355
AW274018 LPAR3 Lysophosphatidic acid receptor 3 3.233
BF432648 TNFRSF19 Tumor necrosis factor receptor superfamily, member 19 3.000
*Threefold changes were detected because this gene was located at two distinct probes on the microarray.
Otomo et al. www.pnas.org/cgi/content/short/1412062112 10 of 10

PDF Document reader online

This website is focused on providing document in readable format, online without need to install any type of software on your computer. If you are using thin client, or are not allowed to install document reader of particular type, this application may come in hand for you. Simply upload your document, and Docureader.top will transform it into readable format in a few seconds. Why choose Docureader.top?

  1. Unlimited sharing - you can upload document of any size. If we are able to convert it into readable format, you have it here - saved for later or immediate reading
  2. Cross-platform - no compromised when reading your document. We support most of modern browers without the need of installing any of external plugins. If your device can oper a browser - then you can read any document on it
  3. Simple uploading - no need to register. Just enter your email, title of document and select the file, we do the rest. Once the document is ready for you, you will receive automatic email from us.

Previous 10

Next 10