1. Volume 21, Issue 5, Pages 1227-1239 (October 2017)
TFCP2 Is Required for YAP-Dependent Transcription to Stimulate Liver Malignancy 
Xiao Zhang, Fenyong Sun, Yongxia Qiao, Weisheng Zheng, Ya Liu, Yan Chen, Qi Wu, Xiangfan Liu, Guoqing Zhu, Yuxin Chen, Yongchun Yu, Qiuhui Pan, Jiayi Wang 
Cell Reports 
Volume 21, Issue 5, Pages (October 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 1227-1239DOI: (10.1016/j.celrep.2017.10.017)
Copyright © 2017 The Authors Terms and Conditions

3. Figure 1 Close Correlation between YAP and TFCP2
(A) MS analysis of YAP binding proteins. The YAP protein was immunoprecipitated by an anti-YAP antibody and analyzed by ESI-Q-TOF-MS three independent times.
(B) Interactions between YAP and TFCP2. Purified YAP or TFCP2 proteins were incubated at 37°C for 90 min before immunoprecipitation and analysis with the indicated antibodies (upper left). Co-localization of YAP and TFCP2 in Bel-7402 and SMMC-7721 cells as measured by confocal microscopy. Scale bar, 20 μm (upper right). Direct interaction between YAP and TFCP2 as measured by PLA. The areas with rectangle boxes were enlarged at the right side. Arrows indicate direct interactions. Scale bar, 100 μm (lower).
(C) TFCP2 expression in liver cancer. YAP and TFCP2 were analyzed by WB (left) and IHC (right), respectively, in patients with liver cancer. The liver tumors and adjacent normal liver tissue samples were paired (right). Scale bar, 500 μm.
(D) TMA of YAP and TFCP2 in liver cancer. Representative IHC images are shown on the left. The data were analyzed using the χ2 test and are shown on the right. Scale bar, 200 μm.
Representative images are shown from three biological replicates, with the exception of TMA.
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4. Figure 2 TFCP2 Is Required for YAP-Induced Malignancy
(A) Expression of the TFCP2 protein under the indicated conditions was measured by WB.
(B) TFCP2 knockdown-mediated impairments in transformed phenotypes were reversed by TFCP2 overexpression in liver cancer cells. Levels of cell viability, colony formation capacity, and caspase-3/7 activity were measured using an MTT-based assay (left), a soft-agar colony formation assay (middle), and Caspase 3/7 Glo LUC Reagent (right), respectively.
(C) TFCP2 silencing inhibited tumor growth in vivo. TFCP2 levels in xenografts were measured by WB (left). n = 5 per group. The xenograft image is shown on the left, and the data and graft are shown on the right.
(D) YAP-stimulated transformed phenotypes were blocked by TFCP2 knockdown in hepatocytes. Cell viability, caspase-3/7 activity, and colony formation capacity were measured using an MTT-based assay (upper left), Caspase 3/7 Glo LUC Reagent (lower left), and a soft-agar colony formation assay (right), respectively.
The data are shown as the means + SD from three biological replicates. ∗∗p < The data shown in (B) and (D) were analyzed using a one-way ANOVA test. The data shown in (C) were analyzed using a two-way ANOVA test.
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5. Figure 3
TFCP2 Synergizes with YAP to Induce Liver Growth, Possibly via a WW-PSY Interaction
(A and B) TFCP2 synergistically affects YAP-stimulated liver growth. The indicated expression plasmids (pLIVE-vector as the backbone) were transfected into mice. Representative confocal microscopy images of H&E-stained liver tissues expressing the indicated proteins were measured by confocal microscopy and H&E staining, respectively. Scale bar, 100 μm (A). General views of liver tissues with the same treatment as (A) are shown (B, left), and the liver weights are also graphed (B, right), n = 5 per group.
(C) YAP bound to TFCP2 via both WW domains. The indicated WT or mutant YAP-HA and WT TFCP2-Myc expression plasmids were co-transfected into Bel-7402 cells followed by immunoprecipitation and analysis with the indicated antibodies. Myc levels in each coIP sample were adjusted to the same content.
(D) TFCP2 bound to YAP via the PSY motif. The indicated WT or mutant TFCP2 and WT YAP-HA expression plasmids were co-transfected into Bel-7402 cells followed by immunoprecipitation and analysis with the indicated antibodies. HA levels in each coIP sample were adjusted to the same content.
∗p < The data shown in (B) were analyzed using a one-way ANOVA test.
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6. Figure 4 TFCP2 Prolongs the YAP Half-Life
(A) YAP and CTGF were regulated by TFCP2. The expression of the YAP, CTGF, and TFCP2 proteins in Bel-7402 and SMMC-7721 cells was measured by WB.
(B) TFCP2 overexpression prolonged the YAP half-life. WB of YAP levels in Bel-7402 (left) or SMMC-7721 cells (right) with or without TFCP2 overexpression that were treated with CHX (50 μg/ml) for the indicated times.
(C and D) TFCP2 knockdown decreased the YAP half-life. WB of YAP levels in Bel-7402 (C) or SMMC-7721 cells (D) with or without TFCP2 knockdown that were treated with CHX (50 μg/ml) for the indicated times.
The data are presented as the means + SD from three biological replicates. YAP levels were normalized to GAPDH levels, and the data from the 0-hr time point were arbitrarily set to 100%.
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7. Figure 5
Genes Co-relegated by YAP and TFCP2 Enhance the Transformed Phenotypes in Liver Cancer Cells
(A) ChIP-seq and RNA-seq revealed genes that are co-regulated by YAP and TFCP2.
ChIP-seq was performed in Bel-7402 cells using anti-YAP and anti-TFCP2 antibodies, respectively (left). RNA-seq was performed in Bel-7402 cells with or without YAP or TFCP2 knockdown (right). Five genes that were co-regulated by YAP and TFCP2 were predicted by ChIP-seq and RNA-seq (middle).
(B) Co-occupancies of YAP and TFCP2 were measured by ChIP and Re-ChIP experiments using anti-YAP or anti-TFCP2 antibodies in Bel-7402 cells.
(C) FYB, PDE3A, BICC1, LINC01021, and LOC were co-regulated by YAP and TFCP2. Levels of the FYB, PDE3A, and BICC1 proteins were measured by WB in Bel-7402 and SMMC-7721 cells with or without infection with the indicated shRNA (left). Levels of the LINC01021 and the LOC mRNAs were measured by qRT-PCR following the above treatments (right).
(D) Knockdown of genes co-regulated by YAP and TFCP2 impaired the transformed phenotypes. Cell viability, colony formation capacity, and caspase-3/7 activity were measured using an MTT-based assay (left), a soft-agar colony formation assay (middle), and Caspase 3/7 Glo LUC Reagent (right), respectively, in cells infected with or without the indicated shRNAs.
The data are presented as the means + SD from three biological replicates. ∗∗p < The data shown in (C) and (D) were analyzed using a one-way ANOVA test. P, peak.
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8. Figure 6
TFCP2 Synergistically Affects YAP-Stimulated Transcription Factor Activity
(A) De novo motif analysis of overlapping YAP and TFCP2 binding sites.
(B) Location of transcription factor binding sites within the peaks predicted by ChIP-seq.
(C) Activation of peaks by YAP and TFCP2 relies on transcription factor binding sites. Activities from peaks with or without indicated transcription factor binding sites in the presence or absence of YAP or TFCP2 overexpression in Bel-7402 cells were measured using the Dual-Luciferase reagent.
(D) YAP bound to predicted transcription factors. Proteins were immunoprecipitated by anti-YAP antibodies, and co-immunoprecipitation of the indicated transcription factors in Bel-7402 or SMMC-7721 cells was measured by WB using the indicated antibodies.
(E) TFCP2 stimulated YAP binding to transcription factors. Lysates from Bel-7402 or SMMC-7721 cells with TFCP2 overexpression (left) or knockdown (right) were immunoprecipitated by anti-YAP antibodies, and co-immunoprecipitation of transcription factors was measured by WB using the indicated antibodies. YAP levels in each sample were adjusted to the same content.
The data are presented as the means + SD from three biological replicates. ∗p < 0.05, ∗∗p < The data shown in (C) were analyzed using a one-way ANOVA test. P, peak.
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9. Figure 7
YAP Binds to Its Transcription Factors via the (G/A)N2(S/G)N5P Motif
(A) The predicted conserved motif within the transcription factors.
(B–F) Interactions between YAP and transcription factors rely on the YBF. Bel-7402 or SMMC-7721 cells were co-transfected with plasmids expressing YAP-HA (WT or Del-2WW) and plasmids expressing the indicated transcription factors (B, POU3F2-WT-FLAG or POU3F2-Mut-FLAG; C, FOXA1-WT-FLAG or FOXA1-Mut-FLAG; D, FOXC1-WT-FLAG or FOXC1-Mut-FLAG; E, MSC-WT-FLAG or MSC-Mut-FLAG; F, NR1D1-WT-FLAG or NR1D1-Mut-FLAG). The exogenous transcription factors were immunoprecipitated by anti-FLAG antibodies, and co-immunoprecipitations of YAP-HA were measured by WB using anti-HA antibodies. FLAG levels in each coIP sample were adjusted to the same content.
(G) Schematic of a possible mechanism underlying the ability of the transcription complex comprising YAP and TFCP2 to stimulate liver tumorigenesis.
Representative images from three biological replicates are shown.
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