YAP inhibits NF-κB signaling and ccRCC growth by opposing p65-ZHX2 cooperativity

Hippo pathway functions as a tumor suppressor pathway by inhibiting the oncogenic potential of pathway effectors YAP/TAZ. However, YAP can also function as a context-dependent tumor suppressor in several types of cancer including clear cell renal cell carcinomas (ccRCC). Here we show that YAP blocks NF-κB signaling in ccRCC to inhibit cancer cell growth. Mechanistically, YAP inhibits the expression of ZHX2, a critical p65 co-factor in ccRCC. Furthermore, YAP competes with ZHX2 for binding to p65. Consequently, elevated nuclear YAP blocks the cooperativity between ZHX2 and p65, leading to diminished NF-κB target gene expression. Pharmacological inhibition of Hippo/MST1/2 blocked NF-κB transcriptional program and suppressed ccRCC cancer cell growth, which can be rescued by ZHX2/p65 overexpression. Our study uncovers a novel crosstalk between the Hippo and NF-κB pathways and its involvement in ccRCC growth inhibition, suggesting that targeting the Hippo pathway may provide a therapeutical opportunity for ccRCC treatment.


Introduction
Initially discovered in Drosophila, the Hippo tumor suppressor pathway is an evolutionarily conserved signaling pathway that controls organ size, tissue homeostasis, and cancer progression among different species [1][2][3] .The Hippo pathway consists of a core kinase cascade with upstream kinases Hippo (Hpo)/MST1/2 phosphorylating and activating downstream kinases LATS1/2 [4][5][6][7][8] , resulting in phosphorylation of the transcriptional effector proteins YAP/TAZ, which inhibits YAP/TAZ activity by restricting their nuclear translocation [9][10][11] .When the activity of Hippo kinase cascade is compromised, unphosphorylated YAP/TAZ translocates into the nucleus, forms a complex with the TEAD-family of transcription factors, and activates Hippo pathway target genes [11][12][13] .Aberrant activation of YAP due to mutation in upstream components, gene amplification or fusion, or other mechanisms promotes tumor progression in many types of cancer including Hepatocellular carcinoma, lung adenocarcinoma, gastric cancer, colon cancer, mesothelioma, schwannomas, ependymomas, cervical squamous cell carcinoma, uveal melanomas, and esophageal squamous cell carcinoma 2,3,14 .Consistent with these clinical observations, transgenic overexpression of YAP or knockout of upstream Hippo pathway components in mouse livers resulted in hepatomegaly, leading to hepatocellular carcinoma formation 10,[15][16][17][18][19][20] .Taken together, these studies have led to a prevalent view that Hippo signaling functions as a tumor suppressor pathway by blocking the oncogenic potential of YAP/TAZ.However, recent studies have revealed that YAP can function as a context-dependent tumor suppressor in several types of cancer including hematological cancers 21 , estrogen receptor a (ERa) positive breast cancer 22,23,24 , androgen receptor (AR) positive prostate cancer 25 , and VHL negative clear cell renal cell carcinoma (ccRCC) 26 .ccRCC makes up ~80% of kidney cancer, which is among the top ten most diagnosed cancers around the world 27 .ccRCC is responsible for most of the kidney cancer-associated death 28 .The majority cases of ccRCC (>90%) resulted from loss-of-function the Von Hippel-Lindau (VHL) tumor suppressor gene, leading to stabilization and activation of Hypoxiainducible factor 2α (HIF-2α) transcriptional program 29 .In addition to regulating HIF-2α, the VHL E3 ubiquitin ligase has other substrates that may also play important role in the progression of ccRCC because ccRCC patient samples exhibited differential sensitivity to HIF-2α inhibitors [30][31][32] .
A genome-wide in vitro expression strategy identified Zinc fingers and homeoboxes 2 (ZHX2) as a VHL substrate 33 .ZHX2 plays a pivotal role in ccRCC progression by activating the nuclear factor kB (NF-kB) pathway via interacting with the p65 subunit of NF-kB 33 .
Our previous study revealed that inhibition of Hippo signaling pathway or transgenic activation of YAP blocked the HIF2 transcriptional program and ccRCC tumor growth 26 .Here, we showed that YAP activation also inhibited NF-kB pathway in addition to HIF-2.
Mechanistically, YAP acts in conjunction with TEAD to inhibit the expression of ZHX2 and its ability to bind p65, thereby blocking the cooperativity between ZHX2 and p65 required for NF-kB target gene expression and ccRCC growth.Pharmacological inhibition of Hippo/MST1/2 kinase activity inhibited p65/ZHX2 cooperativity, leading to diminished NF-kB target gene expression and inhibited ccRCC tumor growth, which can be alleviated by increasing the activity of ZHX2 or p65.

YAP activation inhibits ccRCC cancer cell growth in 2D cultures
We have previously shown that activation of YAP, either by treatment with a small molecule inhibitor of Hippo/MST1/2 kinase XMU-MP-1 34 , or by overexpression of a constitutively active YAP (YAP-5SA) 9 , inhibited ccRCC cell growth in both 3D cultures and Xenografts 26 .Consistent with growth inhibition, both XMU-MP-1 and YAP5SA inhibit HIF-2 pathway activity, which is required for ccRCC cell growth in both 3D cultures and mice.Intriguingly, we found that XMU-MP-1 also inhibited cell growth in 2D cultures of multiple VHL mutant ccRCC cell lines including 786-O cells where HIF-2 pathway activity was dispensable for their growth (Fig. 1a-c) 31,35,36 , raising the possibility that XMU-MP-1 could inhibit ccRCC cell growth through a mechanism(s) other than blocking HIF-2 pathway activity.
To determine whether XMU-MP-1 inhibited ccRCC cell growth in 2D cultures through the Hippo pathway instead of an off-target effect, we generated Flag (Fg)-tagged wild type (Fg-MST2 WT ) and a mutant form of MST2 carrying Y101D and D109A double mutations (Fg-MST2 Y101D/D109A ).It has been demonstrated previously that MST2 Y101D/D109A exhibited normal kinase activity; however, it no longer binds XMU-MP-1 and thus is insensitive to drug inhibition 34 .As expected, expression of either Fg-MST2 WT or Fg-MST2 Y101D/D109A increased the cytoplasmic level while decreased the nuclear level of YAP in 786-O cells, suggesting that exogenously expressed MST2 could increase the phosphorylation of YAP, leading to its cytoplasmic retention (Fig. 1d).Expression of either Fg-MST2 WT or Fg-MST2 Y101D/D109A in 786-O cells slightly increased cell growth (Fig. 1e); However, only Fg-MST2 Y101D/D109A significantly rescued XMU-MP-1-mediated growth inhibition (Fig. 1f).Hence, XMU-MP-1 inhibited 786-O cell growth mainly through an on-target effect, i.e., by inhibiting MST1/2 kinase activity to increase YAP nuclear localization.
To further investigate how perturbation of Hippo signaling affects ccRCC cell growth in 2D culture, we treated 786-O cells with different doses of XMU-MP-1 and found that XMU-MP-1 inhibited 786-O cell growth in a dose dependent manner (Fig. 1g).Furthermore, infection of 786-O cells with lentivirus expressing either the constitutively active YAP (YAP-5SA) or TAZ (TAZ-4SA) resulted in significant growth inhibition (Fig. 1h-i

YAP activation inhibits the transcriptional program of NF-kB in ccRCC
A previous study identified ZHX2 as a VHL target upregulated in VHL mutant ccRCC tumors and showed that ZHX2 is essential for VHL mutant ccRCC cell growth in vitro and in vivo 33 .
Furthermore, this study demonstrated that ZHX2 promoted ccRCC tumor growth by promoting NF-kB pathway activity.
To confirm that Hippo pathway regulates NFkB signaling in ccRCC, we carried out RT-qPCR experiments to measure the change in NFkB target gene expression in ccRCC upon treatment with XMU-MP-1 at different doses or at a fixed concentration for increasing length of time.As shown in Fig. 2d, High dose of XMU-MP-1 resulted in a more dramatic downregulation of NFkB target gene expression.Similarly, increasing the duration of XMU-MP-1 treatment progressively decreased the expression of multiple NF-kB target genes without affecting the expression of p65 protein level (Fig. 2e; Fig. S2).These results suggest that XMU-MP-1 inhibits NFkB target gene expression in a dose-and time-dependent manner and that this downregulation is not due a change in the abundance of p65, a major subunit of NFkB in ccRCC 33 .Activation of YAP by the constitutively active YAP5SA resulted in inhibition of NF-kB target gene expression (Fig. 3a).On the contrary, knockdown of both YAP and TAZ using a previously validate siRNA 25,26 , increased NFkB target gene expression (Fig. 3b).These results suggest that YAP negatively regulates NFkB signaling activity.YAP regulates gene expression through the TEAD family of transcription factors including TEAD1-4 37 .RNAseq analysis revealed that TEAD1 and TEAD4 were more abundantly expressed than TEAD2 and TEAD3 in 786-O cells 26 .Overexpression of either TEAD1 or TEAD4 downregulated NF-kB target gene expression whereas knockdown of TEAD1/3/4 using previously validate siRNAs that targets shared sequences among these TEADs 12,25 , led to increased expression of NF-kB target genes (Fig. 3c-d).These results supporting the notion that YAP acts in conjunction with TEAD to inhibit NFkB target gene expression.

YAP inhibits ZHX2 expression
Analysis of the RNAseq data revealed that XMU-MP-1 treatment of 786-O cells also downregulated the expression of ZHX2.By RT-qPCR, we confirmed that MST1/2 inhibition by XMU-MP-1 decreased the expression of ZHX2 in 786-O cells (Fig. 2f).Expression of YAP-5SA also downregulated whereas knockdown of YAP/TAZ increased ZHX2 expression in 786-O cells (Fig. 3e-f), suggesting that YAP inhibits ZHX2 expression.
Because ZHX2 is a critical cofactor for p65 in the regulation of NFkB target gene expression, YAP-mediated downregulation of ZHX2 could explain why XMU-MP-1 and YAP-5SA inhibit the expression of NFkB target genes.However, in time course experiments, we found that XMU-MP-1 inhibited NFkB target genes as well as ZHX2 within 8 hours whereas ZHX2 protein levels were noticeably downregulated only after 16 hours treatment (Fig. S3a-c).
Whereas transcriptional downregulation of ZHX2 may contribute to a long-term shutdown of NFkB target gene expression by YAP activation, the immediate response of NF-kB target genes to XMU-MP-1 treatment is likely due to a differently mechanism(s).

YAP blocks the interaction between ZHX2 and p65
Because ZHX2 promotes NFkB target gene expression by physically interacting with p65, we carried out co-immunoprecipitation (Co-IP) experiments to determine whether YAP inhibits NFkB pathway activity by inhibiting p65/ZHX2 association.Using antibodies against endogenous proteins, we found that p65 formed a complex with ZHX2 as well as YAP and TEAD4 (Fig. 4a).Interestingly, XMU-MP-1 decreased the amount of ZHX2 associated with p65 while simultaneously increased the amount of YAP and TEAD4 bound to p65 (Fig. 4a), suggesting that YAP-TEAD may compete with ZHX2 for binding to p65.
To further characterize the competition between YAP-TEAD and ZHX2 for p65 binding, HEK293 cells were transfected with fixed amounts of Myc-tagged p65 (Myc-p65) and HAtagged ZHX2 (HA-ZHX2) and increasing amounts of Flag-tagged YAP-5SA (Fg-YAP-5SA) or GFP-tagged TEAD4 (TEAD4-GFP), followed by Co-IP and western blot analysis.As shown in Fig. 4b, increasing the expression level of Fg-YAP-5SA progressively increased the amount of p65/Fg-YAP-5SA complex with concomitant decrease in the amount of p65-ZHX2 complex.Furthermore, increasing the amount of Fg-YAP-5SA also resulted in an increase in the amount endogenous TEAD4 associated with Myc-p65.Similarly, increasing the amount of TEAD4-GFP led to a decrease in the amount of HA-ZHX2 associated with Myc-p65 but an increase in the amounts of TEAD4--GFP and endogenous YAP associated with Myc-p65.These results suggest that binding of YAP and TEAD to p65 disrupt the formation of the p65-ZHX2 complex.
In a reciprocal experiment, HEK293 cells were transfected with fixed amounts of Myc-p65, Fg-YAP5SA and TEAD4-GFP, and increasing amount of HA-ZHX2.As shown in Fig. 4d, increasing the expression level of HA-ZHX2 progressively increased the amount of p65-ZHX2 complex with concomitant decrease in the amounts of Fg-YAP5SA and TEAD4-GFP associated with Myc-p65.Taken together, these results demonstrate that YAP-TEAD and ZHX2 compete for binding to p65.

YAP impedes binding of p65 and ZHX2 to NF-kB target promoters
ZHX2 and p65 co-occupy on the promoter/enhancer regions of many NFkB target genes and ZHX2/p65 interaction facilitates their promoter/enhancer occupancy to NF-kB target genes as knockdown of ZHX2 impaired p65 binding to IL6 and IKBKE promoters, and vice versa 33 .Therefore, we tested whether YAP activation by Hippo pathway inhibition reduces the binding of p65/ZHX2 to the promoter regions of their coregulated genes by carrying ChIP-qPCR experiments.As shown in Fig. 5, after 786-O cells were treated with XMU-MP-1, both ZHX2 and p65 exhibited decreased chromatin association at multiple NF-kB target promoters cobound by p65 and ZHX2, including IL6, IL8, and CCL2 (Fig. 5a-c).Interestingly, p65 and ZHX2 co-occupy the promoter region of ZHX2, and their chromatin association was reduced by XMU-MP-1 treatment (Fig. 5a-c), suggesting that ZHX2 is an autoregulated NF-kB target gene and that YAP inhibits ZHX2 expression by disrupting p65/ZHX2 interaction.

ZHX2/p65 overexpression rescued ccRCC growth inhibited by XMU-MP-1
If Yap activation inhibits ccRCC cancer cell growth in 2D cultures by impeding p65/ZHX2 cooperativity, one would predict that increasing the activity of either ZHX2 or p65 may rescue ccRCC cancer cell growth inhibited by XMU-MP-1.To test this possibility, we overexpressed ZHX2 or p65 in 786-O cells via lentiviral infection and measure cancer cell growth in 2D cultures as well as NF-kB target gene expression in the absence or presence of XMU-MP-1 treatment.
As expected, overexpression of either ZHX2 or p65 increased NF-kB target gene expression and promoted ccRCC cell growth in the absence of drug treatment (Fig. 6a-f).Importantly, ZHX2 or p65 overexpression significantly rescued ccRCC cell growth and NF-kB target gene expression after XMU-MP-1 treatment (Fig. 6a-f), lending further support that YAP inhibits ccRCC cancer cell growth by opposing the cooperative activity of p65 and ZHX2.

Discussion
Despite the prevailing view that Hippo signaling inhibits tumor growth by blocking the oncogenic potential of YAP/TAZ 2,14 , recent studies revealed that YAP can act as a context-dependent tumor suppressor in several types of cancer [21][22][23][24][25][26]38,39 . Our pevious study uncovered a noncanonical Hippo signaling mechanism in ccRCC whereby TEAD functions as a critical cofactor for the ccRCC oncogenic driver HIF-2α whereas nuclear YAP inhibits HIF-2α signaling by competing with HIF-2α for TEAD 26 .Analogous mechanisms have also been proposed to account for the tumor suppressor function of YAP in ERa + breast cancer and AR + prostate cancer 23 25 .In the current study, we uncovered yet another mechanism by which YAP inhibits cancer cell growth, i.e., through inhibiting the NFkB signaling pathway.
Previous studies showed that the growth of VHL -/-ccRCC cancer cells in 2D cultures was independent of HIF-2α activity 31,35,36 .Our observation that XMU-MP-1 could inhibit VHL -/-ccRCC growth not only in soft agar (3D culture) but also in 2D culture, a condition in which HIF-2α is dispensable for 786-O growth, implied that YAP may inhibit VHL -/-ccRCC growth through additional mechanism(s) independent of HIF-2α.Furthermore, such inhibitory mechanism depends on the status of VHL as restoring VHL activity in 786-O cells blunted the inhibitory effect of XMU-MP-1, suggesting that YAP may inhibit 786-O cell growth through another VHL substrate(s).The recent finding that ZHX2 is a VHL substrate that promotes ccRCC cancer progression by forming a complex with p65 to regulate NF-kB target genes let us to speculate that YAP may target the p65-ZHX2 signaling axis.Indeed, we found that treatment of 786-O with XMU-MP-1 downregulated many NF-kB target genes coregulated by p65 and ZHX2 (Fig. S1).XMU-MP-1 treatment disrupted the interaction between p65 and ZHX2 while increasing the interaction between p65 and YAP/TEAD.Co-IP experiments in a heterologous system further demonstrate the competition between YAP/TEAD and ZHX2 for p65 binding.Increasing the activity of ZHX2/p65 largely rescued ccRCC growth inhibited by XMU-MP-1.Based on these and other observations, we propose that YAP inhibits NF-kB signaling in ccRCC by opposing p65/ZHx2 cooperativity, which contributes to ccRCC growth inhibition (Fig. 7).ccRCC is responsible for most of the kidney cancer-associated death.Therapies targeting HIF-2α downstream effectors such as VEGF are the standard of care; however, drug resistance occurs in most patients 32,40 .HIF-2α inhibitors have recently entered the clinic; however, ccRCC patient samples exhibited differential sensitivity to HIF-2α inhibitors and HIF-2α mutants could render drug resistance 30,41 .Our findings that Hippo pathway inhibition or YAP activation not only inhibits HIF2α but also NF-kB signaling in ccRCC open a possibility for developing novel therapeutics to treat ccRCC.
To generate Flag-MST2-dm, Y101F/D109A double mutation was amplified by PCR-based mutagenesis from Flag-MST2 and the product was subcloned to pLVX-ZsGreen vector.The YAP-WT and YAP-5SA constructs were described previously 42

Immunoblot analysis
Cells were harvested and lysed with lysis buffer containing 1M Tris pH8.0, 5M NaCl, 1M NaF, 0.1M Na3VO4, 1% NP-40, 10% glycerol, and 0.5M EDTA (pH 8.0).Proteins were separated by electrophoresis on SDS-polyacrylamide gel electrophoresis (PAGE) and electro-transferred to PVDF membrane.Membranes were washed with TBST and incubated with primary antibodies for 2 hours.And then the membranes were washed for three times with TBST and incubated with second antibodies for 2 hours, after washed for three times with TBST, the membranes were probed with ECL system (Cytiva, Cat.No. RPN2105).The antibodies used in this study were listed here: rabbit anti-p65 (Cell Signaling Technology Cat.No,8242); rabbit anti-ZHX2

Co-immunoprecipitation assay
Immunoprecipitation was performed according to standard protocol.786-O cell lysates were incubated with antibodies or mouse IgG for overnight at 4℃, followed by immobilization and precipitation with Protein A resin (Thermo Scientific Cat.No53139).The bound proteins were analyzed by western blot.For overexpression experiments, HEK293A cells were transfected with 5μg myc-p65, HA-ZHX2, Flag-YAP and GFP-TEAD4 plasmids in 10 cm dishes.Cell lysates were incubated with antibodies against epitope tags for overnight, followed by immobilization and precipitation with Protein A resin.The bound proteins were analyzed by immunoblot assay.

RNA interference
For RNAi experiments in renal cancer cells, the siRNAs were acquired from the Sigma-Aldrich.

RNA extraction and RT-qPCR analysis
We extracted the total RNA by RNeasy plus mini kits according to the protocol (Qiagen, Cat.

ChIP qPCR
ChIP (Chromatin Immuno-precipitation) assay was performed as previously study description 43 , In brief, cells were cross-linked using 2 mM DSG crosslinker (CovaChem, Cat.No.13301) at room temperature for 1 h, followed by secondary fixation with 1% formaldehyde (Pierce, Cat.

RNA sequencing and data analysis
The global gene expression analysis (Vehicle vs XMU-MP-1 treated groups) was based on RNA sequencing platform from BGI (Beijing Genomic Institute).Cellular RNA was extracted using Qiagen RNA extraction kit (Qiagen; Cat: 74104) according to the manufacturer's instructions.
The cellular RNA was sent to BGI Genomics (https://www.bgi.com) for RNA sequencing.RNA was quality-accessed with an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit) with RNA integrity number above 9 for library construction.The total RNA was used for library construction according to the protocol of BGISEQ-500 platform.The libraries were sequenced using BGISEQ-500 platform.Then the FASTQ sequencing files were aligned to the hg19 human genome using STAR aligner with uniquely mapped reads kept for further analysis.Differential expression was analyzed using DESeq2 with default parameters.The RNA sequence data are deposited in the Gene Expression Omnibus (GEO) database (Assessing number: GSE197468).
For gene set enrichment analysis of RNA-seq data, gene sets of HIF2A activated target genes was used and downloaded from Molecular Signatures Database v7.4,GSEA was implemented using the GSEA 4.1.0software, with default parameters.Volcano plot of DEGs (Threshold P<0.01 and fold change>2) was generated using the OmicStudio tools (https://www.omicstudio.cn/tool).

Statistics and reproducibility
All experiments were performed at least three independent times unless noted.Student's t-test was used for comparisons.A P-value of < 0.05 was considered to be significant.Error bars on the graphs were presented as the s.d.When Hippo signaling is "on", YAP is phosphorylated and excluded from the nucleus, allowing p65 and ZHX2 to cooperatively activate NF-kB target gene expression that drives ccRCC growth.When Hippo signaling is "off", YAP enters the nucleus and forms a complex with TEAD.YAP/TEAD impedes p65/ZHX2 cooperativity by disrupting their physical interaction, leading to downregulation of NF-kB target gene expression and growth inhibition.c Western blot analysis of ZHX2 and p65 protein expression in 786-O cells treated with 2 µM XMU-MP-1 for the indicated periods of time.GAPDH was used as a loading control.ZHX2 level started to decline after XMU-MP-1 treatment for 16 hours while p65 protein level remained unchanged even after 24 hours' treatment.
).To determine whether XMU-MP-1 inhibited ccRCC cell growth depending on the status of VHL, we introduced VHL back into 786-O to generate 786-O-VHL cell lines.Compared with original 786-O cell line or a control 786-O cell line expressing the empty vector (786-O-Vector), 786-O-VHL cells are less sensitive to XMU-MP-1-mediated growth inhibition (Fig. 1j-l), suggesting that XMU-MP-1 may act through another VHL target(s) to inhibit 786-O cell growth in 2D cultures.

For
packaging lentivirus, HEK293T cells were transfected with the expression vectors and package vectors (psPAX2 and pMD2.G) by PolyJet (SignaGen laboratories, Cat.No. SL100688).After 48 hours, the supernatants of the medium were collected and filtered with 0.45 μm filter.The supernatant containing virus was stored in 4℃ for cell infection.The ccRCC cells were cultured in fresh media and subsequently infected with lentivirus overnight together with Polybrene (Sigma, Cat.No. H9268).Hygromycin B (Sigma, Cat.No. 10843555001) and Puromycin (Sigma, Cat.No. P9620) were used for infected cells selection according to the resistance of the vectors.
No. 74106).After RNA extraction, the RNA was subjected to reverse transcription PCR for cDNA synthesis according to the RT-PCR kit (Applied Biosystems, Cat.No. 4368814).The relative gene expression was measured according to 2 -ΔΔCT methods.The house keeping gene 36B4 was used for internal control.The Primer sequences were: 36B4, F: GGC GAC CTG GAA GTC CAA CT; R: CCA TCA GCA CCA CAG CCT TC;IL6, F: AGA CAG CCA CTC ACC TCT TCA G, R: TTC TGC CAG TGC CTT TTG CTG; IL8, F: GAG AGT GAT TGA GAG TGG ACC AC, R: CAC AAC CCT CTG CAC CCG ATT T; TNF, F: CTC TTC TGC CTG CTG CAC TTT G; R: ATG GGC TAC AGG CTT GTC ACT C; CCL2, F: AGA ATC ACC AGC AGC AAG TGT CC, R: TCC TGA ACC CAC TTC TGC TTG G; ICAM1, F: AGC GGC TGA CGT GTG CAG TAA T, R: TCT GAG ACC TCT GGC TTC GTC A; VCAM1, F: GAT TCT GTG CCC ACA GTA AGG C, R: TGG TCA CAG AGC CAC CTT CTT G; ZHX2, F: ACA CGG GAC CGA TAT GAT GC, R: TTG GAG GGG GAT AAG GAG GG.

Figure 1 YAP
Figure 1 YAP activation inhibits ccRCC cancer cell growth in 2D cultures

Figure 3
Figure 3 YAPTEAD inhibits NF-kB target gene expression in ccRCC

Figure 4 a
Figure 4 YAP/TEAD competes with ZHX2 for binding to p65 a Western blot analysis of endogenous p65, ZHX2, YAP and TEAD4 immunoprecipitated with

Figure 5
Figure 5 Hippo pathway inhibition impairs occupancy of p65 and ZXH2 on NF-kB target