SUPPLEMENTARY FIGURE LEGENDS
Fig. S1. The relationship between YAP expression and the tumor types and grade. a) YAP
immunosignal positivity in different subtypes of ovarian cancer tissues calculated using all 384
samples. b) YAP immunosignal positivity in different subtypes of ovarian cancer tissues
calculated using only stage I samples. c) YAP immunosignal positivity in tumor tissues of
different grades. Each group represents mean ± SEM (n = 42 for normal control, n = 19 for clear
cell cancer, n = 50 for mucinous cancer, n = 21 for endometrioid cancer, n = 234 for serous
cancer, n = 62 for grade one group, n = 83 for grade 2 group, and n = 115 for grade 3 group).
Groups with same letters are not significantly different from each other (p > 0.05).
Fig. S2. Quantitative data showing effects of YAP on the proliferation and survival of
ovarian cells. a) Percentages of cells that are Ki-67 positive; b) Percentages of cells that are
TUNEL positive; MXIV: HOSE-MXIV cells; YAP: HOSE-YAP cells; YAPS127A: HOSE-YAPS127A
cells. CTRL: DMSO control; VTPF: verteporfin (5 µM). Results are from three independent
experiments. Bars with different letters are significantly different from each other (p < 0.001).
Fig. S3. YAP stimulates ovarian cancer cell proliferation in vivo. Quantitative data showing
the percentage of the Ki67 positive cells in tumor xenografts derived from TOV21G-MXIV
(control), TOV21G-YAP and TOV21G-YAPS127A cells. Bars with different letters are significantly
different from each other (p < 0.01).
Fig. S4. Effect of YAP on the mRNA expression of EGF-like ligands in HOSE cell lines.
mRNA levels of 11 EGF-like ligands (EGF, TGFα, HBEGF, AREG, EREG, EPGN, NRG1, NRG2,
NRG3, NRG4 and BTC) were determined by qPCR in HOSE-MX, HOSE-YAP and HOSEYAPS127A cells. Bars with the same letters are not significantly different with each other (p >
0.05).
Fig. S5. Effect of YAP on the mRNA expression of EGF-like ligands in HOSE cell lines.
mRNA levels of YAP and 4 ERBBs (ERBB1 or EGFR, ERBB2, ERBB3 and ERBB4) were
determined by qPCR in HOSE-MX, HOSE-YAP and HOSE-YAPS127A cells. Bars with the same
letters are not significantly different with each other (p > 0.05).
Fig S6. Effect of YAP on the mRNA expression of EGF-like ligands in KGN cell lines.
mRNA levels of 11 EGF-like ligands (EGF, TGFα, HBEGF, AREG, EREG, EPGN, NRG1, NRG2,
NRG3, NRG4 and BTC ) were determined by qPCR in KGN-MX, KGN-YAP and KGN-YAPS127A
cells. Bars with the same letters are not significantly different with each other (p > 0.05).
Fig. S7. Effect of YAP on the mRNA expression of EGF-like ligands in KGN cell lines.
mRNA levels of YAP and 4 ERBBs (ERBB1 or EGFR, ERBB2, ERBB3 and ERBB4) were
determined by qPCR in KGN-MX, KGN-YAP and KGN-YAPS127A cells. Bars with the same
letters are not significantly different with each other (p > 0.05).
Fig. S8. YAP regulates expression of ERBB receptors in KGN cells. a) Detection of ErbB1
and ErbB3 protein levels in KGN-MXIV, KGN-YAP and KGN-YAPS127A cells using western blot.
b) Determine the protein levels of ErbB1 and ErbB3 in KGN cells after knockdown of YAP using
YAP siRNA (si-YAP). siGLO (a non-targeting siRNA) was used as a negative control. All
experiments were repeated independently three times and representative images were
presented.
Fig. S9. Effect of YAP on the production of HBEGF and NRG1-β1 in the cultured KGN
cells. KGN-MXIV, KGN-YAP and KGN-YAPS127A cells were incubated in serum free medium for
48h in a 3D hanging drop culture system. The concentrations of HBEGF and NRG1-β1 in the
medium were determined by ELISA. a) the concentrations of HBEGF in 3D medium; b) the
concentrations of HBEGF in conditioned medium. Overexpression of YAP or constitutively
active YAP in KGN and HOSE cells significantly increased the concentrations of HBEGF and
NRG1-β1 in culture medium. Bars with different letters are significantly different from each other
(p > 0.05).
Fig. S10. Disruption of the YAP-TEAD complex suppresses YAP-induced increase in the
expression of ERBB3 and EGF-like ligands. a) The mRNA levels of YAP, ERBB1, ERBB3,
HBEGF, NRG1 and NRG2 before and after verteporfin treatment (72h) were determined using
RT-PCR. b) HBEGF and NRG1 concentrations before and after verteporfin treatment (72h) in
2D-culture medium of TOV21G cells. c & d) HBEGF and NRG1 concentrations in medium of
HOSE-MXIV, HOSE-YAP & HOSE-YAPS127A cells incubated in a 3D-culture system in the
presence or absence of 5 µM verteporfin. HBEGF and NRG1 concentrations were measured by
using ELISA kits. Results are from three independent experiments. Bars with different letters are
significantly different from each other (p < 0.001).
Fig. S11. ERBB3 and HBEGF siRNAs effectively reduce YAP induced ERBB3 and HBEGF
expressions in HOSE and TOV21G cell lines. a) mRNA levels of YAP and ERBB3 before and
after knockdown of ERBB3 with ERBB3 siRNA (siERBB3) in in TOV21G cell lines (top panel)
and in HOSE cell lines (lower panel). b) mRNA levels of YAP and HBEGF before and after
knockdown of HBEGF with HBEGF siRNA (siHBEGF) in TOV21G cell lines (top panel) and in
HOSE cell lines (lower panel). Each bar in bar graphs represents mean ± SEM. Bars with
different letters are significantly different from each other (p < 0.05).
Fig.
S12.
Quantitative
data
showing
that
HBEGF
and
NRG1β1
transiently
dephosphorylate the major components of the Hippo/YAP signaling cascade. The
Western blots in Fig. 9a are quantified with a UVP gel documentation system. The intensity of
the immunosignals were normalized to β-actin. The data is presented as the ratio relative to the
control (0 minutes group). Each point in the graph represents the mean ± SEM (n ≥ 3). : p <
0.05; : p < 0.01; : p < 0.001.
Fig. S13. HBEGF regulates multiple signaling pathways in ovarian cancer cells.
Representative Western blot images showing that HBEGF (50 ng/ml) rapidly stimulates ERBB
phosphorylation and activation of the PI3K/AKT and MAPK pathways in TOV21G ovarian
cancer cells.
Fig. S14. NRG1 regulates multiple signaling pathways in ovarian cancer cells.
Representative Western blot images showing that NRG1-β1 (50 ng/ml) rapidly stimulates ERBB
phosphorylation and activation of the PI3K/AKT and MAPK pathways in in TOV21G ovarian
cancer cells.
Fig. S15. Involvement of PI3K and MAPK signaling pathways in the regulation of YAP
activity by HBEGF and NRG1-β1.
TOV21G cells were pre-treated with pathway specific
inhibitors for 3 hours before being treated with 50 ng/ml HBEGF or NRG1-β1 for 60 min. a)
Representative images showing that EGFR kinase inhibior AG1478 (AG), PI3K inhibitor
LY294002 (LY) and MEK1/2 inhibitor U0126 (UO) totally blocked HBEGF-induced
dephosphorylation of YAP at serine 127. The AKT inhibitor (AKTi-VIII, 5 µM) only partially
blocked HBEGF action. b) Representative images showing that blockade of NRG1-β1-induced
dephosphorylation of YAP protein by LY294004 and UO in TOV21G cells. c) Quantitative data
showing the relative intensity of the immunosignal in panel A). d) Quantitative data showing the
relative intensity of the immunosignal in panel B). Quantitative results are from three
independent experiments. Bars with different letters are significantly different from each other
(p > 0.05).
Fig. S16. Involvement of PI3K and MAPK signaling pathways in the regulation of YAP
activity by HBEGF and NRG1-β1. HOSE-MX, HOSE-YAP and HOSE-YAPS127A cells were
seeded on soft agar in 6-well plates and incubated at 37 C with or without LY294002, UO126
and Verteporfin for 8 days. Representative images showing that PI3K inhibitor (LY294002)
almost eliminates YAP-induced colony formation in the HOSE cells, while MEK1/2 Inhibitor
(UO126) only partially blocked YAP-induced colony formation in HOSE cells. Note that
treatment with verteporfin (5 µM) totally blocked YAP-induced colony formation in HOSE cells.
Scale bar: 1mm.
Fig. S17. Effect of HBEGF and NRG1 on mRNA expression of EGF-like ligands and ERBB
receptors. a) Representative images showing mRNA expression of YAP, EGFR, ERBB3,
NRG1, NRG2 and HBEGF in TOV21G cells treated with or without HBEGF or NRG1 for 48h
and detected by RT-PCR. GAPDH was used as a internal control. b-g) Quantitative results
showing the relative mRNA levels normalized by mRNA levels of the control groups in a). Data
are from three independent experiments. Bars with different letters are significantly different
from each other (p < 0.05).
Fig. S18. Examining the efficiency of the additional siRNA. a) left panel: representative
images showing that the second YAP siRNA (siYAP (2)) successfully knocked down YAP
protein in TOV21G cells; right panel: Knockdown of YAP with siYAP (2) significantly suppressed
TOV21G ovarian cancer cell proliferation. b) Top panel: mRNA levels of YAP and HBEGF
before and after knockdown (72h) of HBEGF with the second HBEGF siRNA (siHBEGF(2)) in
KGN-MXIV, KGN-YAP & KGN-YAPS127A cells; lower panel: mRNA levels of YAP and ERBB3
before and after knockdown (72h) of ERBB3 with the second ERBB3 siRNA (siERBB3(2)) in
KGN-MXIV, KGN-YAP and KGN-YAPS127A cells. c) Knockdown of ERBB3 with siERBB3(2)
totally blocked YAP-induced proliferation of KGN cells (p < 0.0001), while knockdown of HBEGF
with siHBEGF(2) partially but significantly blocked YAP-stimulated KGN cell proliferation (p <
0.05). Bars with different letters are significantly different from each other.