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Figures and figure legends
FIGURE 1. Knockdown of cell-derived connective tissue growth factor (CTGF) reduces
malignant mesothelioma (MM) cell proliferation rate and expression of bone
morphogenetic protein and activin membrane-bound inhibitor (BAMBI).
A, All 7 MM cell lines examined (NCI-H28, MSTO-211H, Y-MESO-8D, Y-MESO-14,
Y-MESO-27,
NCI-H2052,
and
NCI-H2452)
demonstrated
detectable
CTGF
immunoexpression, with highest expression in MSTO-211H, Y-MESO-27, and Y-MESO-14
cell lines. Lines Y-MESO-27 and Y-MESO14 were used primarily for further analyses.
B, Transfection with a CTGF-targeted siRNA (20 nM) reduced CTGF expression at both the
mRNA level (upper panels) as measured by qRT-PCR and at the protein level (low panels) as
measured by western blotting in Y-MESO-14 and Y-MESO-27 cells. Total mRNAs and
soluble proteins were extracted after 48 hours and 72 hours of transfection, respectively.
C, CTGF knockdown reduced Y-MESO-14 and Y-MESO-27 cell proliferation rates as
measured by viable cell counting assay. Cultures were prepared for analysis after 0, 24, 48,
and 72 hours of CTGF siRNA transfection.
D, CTGF knockdown reduced BAMBI expression at both the mRNA level (upper panel) and
the protein level (below).
(B, D, E) Results are expressed as mean ± SD of three independent experiments (*p < 0.05
and **p < 0.01 vs. control siRNA group by one-way ANOVA with post hoc Bonferroni tests
for pair-wise comparisons).
FIGURE 2. Knockdown of BAMBI suppresses MM cell proliferation and enhances
CTGF expression.
A, Transfection of a BAMBI siRNA (20 nM) reduced BAMBI mRNA expression (upper
panels) and protein expression (lower panels) in Y-MESO-14 and Y-MESO-27 cell lines.
Total mRNA and soluble proteins were extracted after 48 hours and 72 hours of transfection,
respectively.
B, Estimated numbers of viable Y-MESO-14 and Y-MESO-27 cells at 0, 24, 48, and 72 hours
after BAMBI siRNA transfection. Knockdown of BAMBI significantly reduced viable cell
numbers compared to control siRNA transfection.
C, Images of Y-MESO-14 and Y-MESO-27 cell cultures after 72 h of transfection with
BAMBI siRNA and control siRNA. Scale bar: 200 μm.
D, BAMBI knockdown reduced expression of CTGF mRNA (upper panel) and protein
(below panel) as measured by qRT-PCR and western blotting, respectively.
A, B, and D, Results are expressed as mean ± SD of three independent experiments (*p <
0.05 and **p < 0.01 vs. control siRNA group by one-way ANOVA with post hoc Bonferroni
tests for pair-wise comparisons).
FIGURE 3. Knockdown of CTGF or BAMBI reduces the expression of cell cycle
regulators.
Y-MESO-14 and Y-MESO-27 cells were transfected with 20 nM BAMBI siRNA (A-D) or
CTGF siRNA (E-H), followed by mRNA extraction after 48 hours for qRT-PCR and soluble
proteins after 72 hours for western blotting.
A, C, E, G, mRNA expression levels of cyclin D1, cyclin D3, CDK2, and CDK4 were
estimated by qRT-PCR using suitable primers. Results are expressed as mean ± SD of three
independent experiments (*p < 0.05 by two-tailed Student’s t-test).
B, D, F, H, Cyclin D1, cyclin D3, CDK2, and CDK4 protein expression by mesothelioma
cells following BAMBI or CTGF knockdown. Both cyclin D3 and CDK2 expression levels
were reduced by either BAMBI or CTGF knockdown.
FIGURE 4. Expression and subcellular localization of BAMBI protein in mesothelial
and mesothelioma cell lines.
A, Cell lysates were extracted from seven mesothelioma cell lines and the mesothelial
MeT-5A cell line. BAMBI protein was ubiquitously expressed by all mesothelioma cell lines
but only weakly or below detection limits by MeT-5A cells.
B, Immunofluorescence staining of Y-MESO-27 and MeT-5A cells showing that BAMBI
localizes to the plasma membrane and cytosol. The nuclei were counterstained with DAPI
(blue). The white arrows demarcate BAMBI expression in the cell membrane. Scale bars: 10
μm.
FIGURE 5. Elevated BAMBI expression predicts shorter overall survival of
mesothelioma patients.
A, Kaplan–Meier survival plots were generated by software from the GEPIA webserver using
mRNA sequence data from TCGA and GTEX. The samples were split into a high BAMBI
expression group (n = 54) and a low BAMBI expression group (n = 29). Log-rank test
indicate that overall survival was significantly reduced by high BAMBI expression (p <
0.05).
B, Potential mechanisms for regulation of MM cell proliferation by CTGF and BAMBI.
CTGF knockdown reduces BAMBI expression and suppresses cell growth by downregulating
cell cycle proteins (black arrow). BAMBI regulates CTGF expression (gray arrow) and
further affects cell growth by regulating cyclin D1 expression (dotted arrow).
Supplementary
FIGURE S1. CTGF knockdown reduces BAMBI mRNA expression in MSTO-211H,
NCI-H28, NCI-H2052, and NCI-H2452 cells.
Malignant mesothelioma (MM) cells were transfected with 20 nM CTGF siRNA or control
siRNA. After 48 hours of transfection, total mRNA was extracted to evaluate the expression
of CTGF and BAMBI by qRT-PCR. Results are expressed as mean ± SD of three
independent experiments (*p < 0.05 and **p < 0.01 vs. control siRNA group by one-way
ANOVA and post hoc Bonferroni tests).
Table S1. Target sequences of siRNAs in Smart pool
Smart pool
siRNA
Target Sequence
J-012633-10
5’-ACAAUGACAUCUUUGAAUC-3’
CTGF
J-012633-11
5’-AGGAAGAUGUACGGAGACA-3’
SO-2804612G
J-012633-12
5’-CGAUUAGACUGGACAGCUU-3’
J-012633-13
5’-GAGAGACAUUAACUCAUUA-3’
J-019596-05
5’-AUAAGAGGCUGCAGGAUCA-3’
BAMBI
J-019596-06
5’-UCACGGACACCAUUCCAAA-3’
SO-2889260G
J-019596-07
5’-GAUCGCCACUCCAGCUACA-3’
J-019596-08
5’-GGGGCAGGUUGCAAAGUUA-3’
5’-UGGUUUACAUGUCGACUAA-3’
Control
5’-UGGUUUACAUGUUGUGUGA-3’
SO-2855667G
5’-UGGUUUACAUGUUUUCUGA-3’
5’-UGGUUUACAUGUUUUCCUA-3’
Table S2. Sequences of primers for qRT-PCR
Genes
Sequences for primers
Accession no.
CTGF
5’-CTGCGAGGAGTGGGTGTGT-3’
NM_001901.3
5’-GAACAGGCGCTCCACTCTGT-3’
BAMBI
5’-CGATGTTCTCTCTCCTCCCAG-3’
NM_012342.3
5’-AATCAGCCCTCCAGCAATGG-3’
Cyclin D1
5’-GCTCCTGGTGAACAAGCTCAA-3’
NM_053056.3
5’-ATGGAGGGCGGATTGGAA-3’
Cyclin D3
5’-GGGACCTGGCTGCTGTGAT-3’
NM_001136017.3
5’-GCGGGTACATGGCAAAGGTA-3’
CDK2
5’-TGTGGTACCGAGCTCCTGAAA-3’
NM_001798.5
5’-AGATCCGGAAGAGCTGGTCAA-3’
CDK4
5’-CCGAGCTCCCGAAGTTCTTC-3’
NM_000075.4
5’-GCAGCCCAATCAGGTCAAAG-3’
GAPDH
5’-CTCTGCCCCCTCTGCTGAT-3’
5’-CAGTCTTCTGGGTGGCAGTGA-3’
NM_002046.7
...
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