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3xflag pcmv5 topo taz s89a (Addgene inc)

Name: 3xflag pcmv5 topo taz s89a
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Addgene inc 3xflag pcmv5 topo taz s89a

Human TAZ/WWTR1 gene promoter is responsive to canonical TGF-β/SMAD pathway. ( A) The pGL3 constructs containing the different DNA fragments of the hTAZ promoter and one mutant are shown. ( B) HepG2 cells were transiently transfected with hTAZ(1.13)-Luc along with ALK5(WT) or ALK5(TD) for 48 h, and then treated or not for 24 h with 0.2 nM TGF-β. At 72 h post-transfection, luciferase activity was measured. Data are representative of 2 independent experiments in triplicate. ( C) HepG2 cells were transiently co-transfected with hTAZ(1.13)-Luc or hTAZ(407)-Luc reporters along with ALK5(WT) or ALK5(TD), and then luciferase activity was measured at 72 h post-transfection; values are expressed as means ± SEM of 2 independent experiments in sextuplicate. ( D) HepG2 cells were transiently transfected with hTAZ(1.13)-Luc or hTAZ(1.13-mutTRE)-Luc constructs, each in co-transfection with ALK5(WT) or ALK5(TD), and luciferase activity was measured at 72 h post-transfection; values are expressed as means ± SEM of 2 independent experiments in sextuplicate. P < 0.05*. ( E) HepG2 cells were co-transfected as indicated, with pGL3-basic/hTAZ(1.13)-Luc along with plasmids bearing SMADs full-length cDNA: <t>pCMV5/Flag-SMAD2</t> (S2), pCMV5/Flag-SMAD3 (S3) or pCMV5/HA-SMAD4 (S4), and ALK5(WT) or ALK5(TD). Then, cells were lysed after 72 h post-transfection, and luciferase activity was analyzed. Raw RLU (Relative Light Units) values are expressed as means ± SEM of 2 independent experiments in quadruplicate. ( F) HepG2 cells were transiently co-transfected with hTAZ(1.13)-Luc alone or with ALK5 (TD); then, ChIP on plasmid assay was carried out using anti-SMAD2 for IP. PCR was performed using primers spanning the canonical SBE region (648 bp) and a part of the pGL3-basic vector (supplementary raw data). Data are representative of 3 independent experiments. ( G) HepG2 cells were transiently transfected without or with ALK5(TD) for indicated times, and then TAZ, pSMAD2, and SMAD2 protein levels were evaluated by immunoblot; β-ACTIN was used as a loading control (supplementary raw data). Data are representative of 2 independent experiments.

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1) Product Images from "TGF-β/SMAD canonical pathway induces the expression of transcriptional cofactor TAZ in liver cancer cells"

Article Title: TGF-β/SMAD canonical pathway induces the expression of transcriptional cofactor TAZ in liver cancer cells

Journal: Heliyon

doi: 10.1016/j.heliyon.2023.e21519

Figure Legend Snippet: Human TAZ/WWTR1 gene promoter is responsive to canonical TGF-β/SMAD pathway. ( A) The pGL3 constructs containing the different DNA fragments of the hTAZ promoter and one mutant are shown. ( B) HepG2 cells were transiently transfected with hTAZ(1.13)-Luc along with ALK5(WT) or ALK5(TD) for 48 h, and then treated or not for 24 h with 0.2 nM TGF-β. At 72 h post-transfection, luciferase activity was measured. Data are representative of 2 independent experiments in triplicate. ( C) HepG2 cells were transiently co-transfected with hTAZ(1.13)-Luc or hTAZ(407)-Luc reporters along with ALK5(WT) or ALK5(TD), and then luciferase activity was measured at 72 h post-transfection; values are expressed as means ± SEM of 2 independent experiments in sextuplicate. ( D) HepG2 cells were transiently transfected with hTAZ(1.13)-Luc or hTAZ(1.13-mutTRE)-Luc constructs, each in co-transfection with ALK5(WT) or ALK5(TD), and luciferase activity was measured at 72 h post-transfection; values are expressed as means ± SEM of 2 independent experiments in sextuplicate. P < 0.05*. ( E) HepG2 cells were co-transfected as indicated, with pGL3-basic/hTAZ(1.13)-Luc along with plasmids bearing SMADs full-length cDNA: pCMV5/Flag-SMAD2 (S2), pCMV5/Flag-SMAD3 (S3) or pCMV5/HA-SMAD4 (S4), and ALK5(WT) or ALK5(TD). Then, cells were lysed after 72 h post-transfection, and luciferase activity was analyzed. Raw RLU (Relative Light Units) values are expressed as means ± SEM of 2 independent experiments in quadruplicate. ( F) HepG2 cells were transiently co-transfected with hTAZ(1.13)-Luc alone or with ALK5 (TD); then, ChIP on plasmid assay was carried out using anti-SMAD2 for IP. PCR was performed using primers spanning the canonical SBE region (648 bp) and a part of the pGL3-basic vector (supplementary raw data). Data are representative of 3 independent experiments. ( G) HepG2 cells were transiently transfected without or with ALK5(TD) for indicated times, and then TAZ, pSMAD2, and SMAD2 protein levels were evaluated by immunoblot; β-ACTIN was used as a loading control (supplementary raw data). Data are representative of 2 independent experiments.

Techniques Used: Construct, Mutagenesis, Transfection, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Western Blot

TGF-β/SMAD and TAZ share target genes. ( A ) HepG2 cells were treated with 0.3 nM TGF-β for indicated times. Total RNA was isolated and mRNA levels of CYR61 were analyzed by RT-PCR with specific primers. β-ACTIN was a control (supplementary raw data). ( B ) The graph shows the densitometry values expressed as means ± SEM of 3 independent experiments; P < 0.05*. ( C ) HepG2 cells were pre-treated for 0.5 h with or without 10 μM SB43 or 1 μM VP, and then treated with 0.3 nM TGF-β at indicated times. The mRNA levels of CYR61 were analyzed. GAPDH was a control (supplementary raw data). ( D ) The graph shows the densitometry values expressed as means ± SEM of 3 independent experiments; P < 0.05*. ( E) HepG2 cells were transiently transfected for 72 h with hTAZ(1.13)-Luc alone (control) or along with H-RasV12, and then treated for 24 h with 0.3 nM TGF-β. Luciferase activity was measured at 72 h post-transfection; values are expressed as means ± SEM of 2 independent experiments in triplicate. ( F) HepG2 cells were transiently transfected for 72 h with hTAZ(1.13)-Luc alone (control) or with indicated concentrations of TAZ S89A, and then luciferase activity was measured; values are expressed as means ± SEM of 2 independent experiments in quadruplicate.

Figure Legend Snippet: TGF-β/SMAD and TAZ share target genes. ( A ) HepG2 cells were treated with 0.3 nM TGF-β for indicated times. Total RNA was isolated and mRNA levels of CYR61 were analyzed by RT-PCR with specific primers. β-ACTIN was a control (supplementary raw data). ( B ) The graph shows the densitometry values expressed as means ± SEM of 3 independent experiments; P < 0.05*. ( C ) HepG2 cells were pre-treated for 0.5 h with or without 10 μM SB43 or 1 μM VP, and then treated with 0.3 nM TGF-β at indicated times. The mRNA levels of CYR61 were analyzed. GAPDH was a control (supplementary raw data). ( D ) The graph shows the densitometry values expressed as means ± SEM of 3 independent experiments; P < 0.05*. ( E) HepG2 cells were transiently transfected for 72 h with hTAZ(1.13)-Luc alone (control) or along with H-RasV12, and then treated for 24 h with 0.3 nM TGF-β. Luciferase activity was measured at 72 h post-transfection; values are expressed as means ± SEM of 2 independent experiments in triplicate. ( F) HepG2 cells were transiently transfected for 72 h with hTAZ(1.13)-Luc alone (control) or with indicated concentrations of TAZ S89A, and then luciferase activity was measured; values are expressed as means ± SEM of 2 independent experiments in quadruplicate.

Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Transfection, Luciferase, Activity Assay

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Journal: Heliyon

Article Title: TGF-β/SMAD canonical pathway induces the expression of transcriptional cofactor TAZ in liver cancer cells

doi: 10.1016/j.heliyon.2023.e21519

Figure Lengend Snippet: Human TAZ/WWTR1 gene promoter is responsive to canonical TGF-β/SMAD pathway. ( A) The pGL3 constructs containing the different DNA fragments of the hTAZ promoter and one mutant are shown. ( B) HepG2 cells were transiently transfected with hTAZ(1.13)-Luc along with ALK5(WT) or ALK5(TD) for 48 h, and then treated or not for 24 h with 0.2 nM TGF-β. At 72 h post-transfection, luciferase activity was measured. Data are representative of 2 independent experiments in triplicate. ( C) HepG2 cells were transiently co-transfected with hTAZ(1.13)-Luc or hTAZ(407)-Luc reporters along with ALK5(WT) or ALK5(TD), and then luciferase activity was measured at 72 h post-transfection; values are expressed as means ± SEM of 2 independent experiments in sextuplicate. ( D) HepG2 cells were transiently transfected with hTAZ(1.13)-Luc or hTAZ(1.13-mutTRE)-Luc constructs, each in co-transfection with ALK5(WT) or ALK5(TD), and luciferase activity was measured at 72 h post-transfection; values are expressed as means ± SEM of 2 independent experiments in sextuplicate. P < 0.05*. ( E) HepG2 cells were co-transfected as indicated, with pGL3-basic/hTAZ(1.13)-Luc along with plasmids bearing SMADs full-length cDNA: pCMV5/Flag-SMAD2 (S2), pCMV5/Flag-SMAD3 (S3) or pCMV5/HA-SMAD4 (S4), and ALK5(WT) or ALK5(TD). Then, cells were lysed after 72 h post-transfection, and luciferase activity was analyzed. Raw RLU (Relative Light Units) values are expressed as means ± SEM of 2 independent experiments in quadruplicate. ( F) HepG2 cells were transiently co-transfected with hTAZ(1.13)-Luc alone or with ALK5 (TD); then, ChIP on plasmid assay was carried out using anti-SMAD2 for IP. PCR was performed using primers spanning the canonical SBE region (648 bp) and a part of the pGL3-basic vector (supplementary raw data). Data are representative of 3 independent experiments. ( G) HepG2 cells were transiently transfected without or with ALK5(TD) for indicated times, and then TAZ, pSMAD2, and SMAD2 protein levels were evaluated by immunoblot; β-ACTIN was used as a loading control (supplementary raw data). Data are representative of 2 independent experiments.

Article Snippet: HepG2 cells were transiently transfected with each pGL3-basic/hTAZ-Luc reporter plasmids with or without any of the following plasmids: pCMV5/TβRI(WT)-HA (ALK5 wild-type or ALK5(WT)), pCMV5/TβRI(T204D)-HA (constitutively activated ALK5 or ALK5(TD)), pCMV5/Flag-SMAD2 (S2), pCMV5/Flag-SMAD3 (S3), or pCMV5/HA-SMAD4 (S4), or in the presence of 3XFlag-pCMV5-TOPO-TAZ(S89A) (No. 24815) from Addgene [ ] or pBABE-puro-H-RasV12 (No. 9051) from Addgene.

Techniques: Construct, Mutagenesis, Transfection, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Western Blot

Journal: Heliyon

Article Title: TGF-β/SMAD canonical pathway induces the expression of transcriptional cofactor TAZ in liver cancer cells

doi: 10.1016/j.heliyon.2023.e21519

Figure Lengend Snippet: TGF-β/SMAD and TAZ share target genes. ( A ) HepG2 cells were treated with 0.3 nM TGF-β for indicated times. Total RNA was isolated and mRNA levels of CYR61 were analyzed by RT-PCR with specific primers. β-ACTIN was a control (supplementary raw data). ( B ) The graph shows the densitometry values expressed as means ± SEM of 3 independent experiments; P < 0.05*. ( C ) HepG2 cells were pre-treated for 0.5 h with or without 10 μM SB43 or 1 μM VP, and then treated with 0.3 nM TGF-β at indicated times. The mRNA levels of CYR61 were analyzed. GAPDH was a control (supplementary raw data). ( D ) The graph shows the densitometry values expressed as means ± SEM of 3 independent experiments; P < 0.05*. ( E) HepG2 cells were transiently transfected for 72 h with hTAZ(1.13)-Luc alone (control) or along with H-RasV12, and then treated for 24 h with 0.3 nM TGF-β. Luciferase activity was measured at 72 h post-transfection; values are expressed as means ± SEM of 2 independent experiments in triplicate. ( F) HepG2 cells were transiently transfected for 72 h with hTAZ(1.13)-Luc alone (control) or with indicated concentrations of TAZ S89A, and then luciferase activity was measured; values are expressed as means ± SEM of 2 independent experiments in quadruplicate.

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Transfection, Luciferase, Activity Assay

a , Yap / Taz transcript levels in ECs sorted from postnatal day (P) 6 mouse retinas as determined by RNA-seq ( n = 4 independent samples). b , YAP/TAZ immunoblot analysis of ECs isolated from murine brains and lungs. c , Schematic of the Taz tag reporter containing GFP, FLAG and a biotin-labelling peptide. d , Expression of the reporter-tagged TAZ protein in whole lung lysates of wild-type, heterozygous and homozygous Taz tag mice. VEGFR2, endothelial marker. e , Quantification of TAZ subcellular localization in ECs of Taz tag/tag P6 retinas. N, preferentially nuclear; NC, nucleo-cytoplasmic; C, preferentially cytoplasmic ( n = 10 independent samples). f , Images of GFP, ERG and PECAM-labelled P6 retinas derived from Taz tag/tag mice. The grey images (lower panels) show the isolated GFP signal. The small (white) boxed area is shown at higher magnification in the upper right corner. Scale bars, 50 μm. g , Immunolabelling for TAZ, GFP and PECAM in P6 retinas of Taz iEC-GOF ( Pdgfb-CreERT2;Rosa26-Taz S89A fl/fl ) and control (Ctrl; Rosa26-Taz S89A fl/fl ) mice. The grey images (right panels) show the isolated TAZ signal. Scale bar, 100 μm. h , Confocal images of PECAM-labelled P6 mouse retinas of Ctrl and Taz iEC-GOF mice. A, artery; V, vein. Scale bar, 200 μm. i , Quantification of vascular parameters in Ctrl and Taz iEC-GOF mutants as indicated (EC area, n = 16 (Ctrl) and 14 ( Taz iEC-GOF ) independent samples; EC number/field, n = 12 (Ctrl) and 10 ( Taz iEC-GOF ) independent samples; EC proliferation, n = 13 (Ctrl) and 6 ( Taz iEC-GOF ) independent samples). j , ERG and PECAM- labelled retinas at P6 showing a hyperplastic vasculature in Taz iEC-GOF mice. k , Immunofluorescence images of the angiogenic front in P6 retinas of Ctrl and Taz iEC-GOF mice labelled for EdU, ERG and PECAM-. Scale bar, 200 μm. Western blot data in b and d are from the respective experiment, processed in parallel and are representative of three independent experiments. For a , d , e and i , data represent mean ± s.e.m.; two-tailed unpaired t -test was used. ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significant. The numerical data, unprocessed western blots and P values are provided as source data.

Journal: Nature Metabolism

Article Title: A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth

doi: 10.1038/s42255-022-00584-y

Figure Lengend Snippet: a , Yap / Taz transcript levels in ECs sorted from postnatal day (P) 6 mouse retinas as determined by RNA-seq ( n = 4 independent samples). b , YAP/TAZ immunoblot analysis of ECs isolated from murine brains and lungs. c , Schematic of the Taz tag reporter containing GFP, FLAG and a biotin-labelling peptide. d , Expression of the reporter-tagged TAZ protein in whole lung lysates of wild-type, heterozygous and homozygous Taz tag mice. VEGFR2, endothelial marker. e , Quantification of TAZ subcellular localization in ECs of Taz tag/tag P6 retinas. N, preferentially nuclear; NC, nucleo-cytoplasmic; C, preferentially cytoplasmic ( n = 10 independent samples). f , Images of GFP, ERG and PECAM-labelled P6 retinas derived from Taz tag/tag mice. The grey images (lower panels) show the isolated GFP signal. The small (white) boxed area is shown at higher magnification in the upper right corner. Scale bars, 50 μm. g , Immunolabelling for TAZ, GFP and PECAM in P6 retinas of Taz iEC-GOF ( Pdgfb-CreERT2;Rosa26-Taz S89A fl/fl ) and control (Ctrl; Rosa26-Taz S89A fl/fl ) mice. The grey images (right panels) show the isolated TAZ signal. Scale bar, 100 μm. h , Confocal images of PECAM-labelled P6 mouse retinas of Ctrl and Taz iEC-GOF mice. A, artery; V, vein. Scale bar, 200 μm. i , Quantification of vascular parameters in Ctrl and Taz iEC-GOF mutants as indicated (EC area, n = 16 (Ctrl) and 14 ( Taz iEC-GOF ) independent samples; EC number/field, n = 12 (Ctrl) and 10 ( Taz iEC-GOF ) independent samples; EC proliferation, n = 13 (Ctrl) and 6 ( Taz iEC-GOF ) independent samples). j , ERG and PECAM- labelled retinas at P6 showing a hyperplastic vasculature in Taz iEC-GOF mice. k , Immunofluorescence images of the angiogenic front in P6 retinas of Ctrl and Taz iEC-GOF mice labelled for EdU, ERG and PECAM-. Scale bar, 200 μm. Western blot data in b and d are from the respective experiment, processed in parallel and are representative of three independent experiments. For a , d , e and i , data represent mean ± s.e.m.; two-tailed unpaired t -test was used. ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significant. The numerical data, unprocessed western blots and P values are provided as source data.

Article Snippet: Human FLAG-YAP S127A (Addgene, no. 27370) and human FLAG-TAZ S89A (Addgene, no. 24815) , were subcloned into the pLVX-TetOne-Puro vector (Clontech, no. 631847).

Techniques: RNA Sequencing Assay, Western Blot, Isolation, Expressing, Marker, Derivative Assay, Immunofluorescence, Two Tailed Test

a , RNA-seq analysis of YAP and TAZ transcript levels in different human EC cultures (n = 3 independent samples). HAECs, human aortic ECs; HMVECs, human microvascular ECs; HUVECs, human umbilical vein ECs; HDLEC, human dermal lymphatic ECs. b , Immunoblot analysis of YAP and TAZ protein expression in different endothelial subtypes. c , Targeting strategy to generate a Taz ( Wwtr1 ) knock-in allele encoding for a GFP, FLAG, and biotin-labelling peptide tagged TAZ protein. A schematic representation of the wild-type Taz ( Wwtr1 ) locus, the targeting vector, the recombined as well as the excised locus is shown. E5 − 7, exons 5 to 7. Triangles denote loxP sites. Neo, neomycin positive selection cassette. GFB, GFP-FLAG-biotin labelling peptide fusion tag. DTA, diphteria toxin negative selection marker. d , PCR analysis of genomic DNA obtained from wild-type, heterozygote and homozygote Taz tag mice. Lane 1, DNA marker. e , RT-qPCR analysis of the canonical YAP/TAZ target gene Ctgf in retinal ECs derived from wild-type (Ctrl) and homozygous Taz tag/tag mice, showing no expression difference between the two genotypes (n = 2 (Ctrl) and 3 ( Taz tag/tag ) independent samples). f , g , ERG- and PECAM- immunofluorescence staining ( f ) and quantification of vascular parameters ( g ) of P6 Ctrl and Taz tag/tag retinas, revealing no gross difference in vascular morphogenesis between the two genotypes (EC area: n = 4 (Ctrl) and 8 ( Taz tag/tag ) independent samples; Number of ECs: n = 3 (Ctrl) and 9 ( Taz tag/tag ) independent samples). h , Images of the vascular front and plexus in TAZ, ERG and PECAM labelled P6 mouse retinas derived from C57BL/6 wild-type mice. The images in grey (right panels) show the isolated TAZ signal. Western blot data in b are from the respective experiment, processed in parallel, and are representative of at least three independent experiments. For a , e and g , data represent mean ± s.e.m.; two-tailed unpaired t-test. ** P < 0.01; **** P < 0.0001; NS, not significant. The numerical data, unprocessed western blots and P values are provided as source data.

Journal: Nature Metabolism

Article Title: A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth

doi: 10.1038/s42255-022-00584-y

Figure Lengend Snippet: a , RNA-seq analysis of YAP and TAZ transcript levels in different human EC cultures (n = 3 independent samples). HAECs, human aortic ECs; HMVECs, human microvascular ECs; HUVECs, human umbilical vein ECs; HDLEC, human dermal lymphatic ECs. b , Immunoblot analysis of YAP and TAZ protein expression in different endothelial subtypes. c , Targeting strategy to generate a Taz ( Wwtr1 ) knock-in allele encoding for a GFP, FLAG, and biotin-labelling peptide tagged TAZ protein. A schematic representation of the wild-type Taz ( Wwtr1 ) locus, the targeting vector, the recombined as well as the excised locus is shown. E5 − 7, exons 5 to 7. Triangles denote loxP sites. Neo, neomycin positive selection cassette. GFB, GFP-FLAG-biotin labelling peptide fusion tag. DTA, diphteria toxin negative selection marker. d , PCR analysis of genomic DNA obtained from wild-type, heterozygote and homozygote Taz tag mice. Lane 1, DNA marker. e , RT-qPCR analysis of the canonical YAP/TAZ target gene Ctgf in retinal ECs derived from wild-type (Ctrl) and homozygous Taz tag/tag mice, showing no expression difference between the two genotypes (n = 2 (Ctrl) and 3 ( Taz tag/tag ) independent samples). f , g , ERG- and PECAM- immunofluorescence staining ( f ) and quantification of vascular parameters ( g ) of P6 Ctrl and Taz tag/tag retinas, revealing no gross difference in vascular morphogenesis between the two genotypes (EC area: n = 4 (Ctrl) and 8 ( Taz tag/tag ) independent samples; Number of ECs: n = 3 (Ctrl) and 9 ( Taz tag/tag ) independent samples). h , Images of the vascular front and plexus in TAZ, ERG and PECAM labelled P6 mouse retinas derived from C57BL/6 wild-type mice. The images in grey (right panels) show the isolated TAZ signal. Western blot data in b are from the respective experiment, processed in parallel, and are representative of at least three independent experiments. For a , e and g , data represent mean ± s.e.m.; two-tailed unpaired t-test. ** P < 0.01; **** P < 0.0001; NS, not significant. The numerical data, unprocessed western blots and P values are provided as source data.

Article Snippet: Human FLAG-YAP S127A (Addgene, no. 27370) and human FLAG-TAZ S89A (Addgene, no. 24815) , were subcloned into the pLVX-TetOne-Puro vector (Clontech, no. 631847).

Techniques: RNA Sequencing Assay, Western Blot, Expressing, Knock-In, Plasmid Preparation, Selection, Marker, Quantitative RT-PCR, Derivative Assay, Immunofluorescence, Staining, Isolation, Two Tailed Test

a , Schematic representations of the wild-type Rosa26 locus, the targeting vector, the recombined as well as the excised allele are shown. A cassette containing the CAG promoter, a floxed STOP sequence, a cDNA encoding for 3xFLAG-tagged TAZ S89A and IRES-nGFP was inserted into the Rosa26 locus. Triangles denote loxP sites. DTA, diphteria toxin negative selection marker; pA, polyA signal. b , Immunoblot analysis of total lung lysates from control (Ctrl, Rosa26-Taz S89A fl/fl ) and mutant mice ( Taz iEC-GOF , Pdgfb-creERT2;Rosa26-Taz S89A fl/fl ). 4OHT was administered from P1 to P4 and samples analyzed at P6. Arrow heads indicate expression of the FLAG-tagged Taz S89A mutant. c , RT-qPCR analysis of retinal ECs at P6, showing increased expression of the canonical YAP/TAZ target gene Ctgf in Taz iEC-GOF mice when compared to Ctrl (n = 4 independent samples). d , Immunofluorescence staining for GFP, ERG and PECAM in P6 retinas depicting a nuclear GFP signal in PECAM positive vessels of Taz iEC-GOF mutants, which is absent in Ctrl mice. e , Higher magnification images of P6 retinas labelled for EdU, ERG and PECAM showing increased EC proliferation in the Taz iEC-GOF mice. f , g , Confocal images ( f ) and quantification of endothelial coverage ( g ) in PECAM labelled P6 retinas obtained from Ctrl ( Yap fl/fl ;Taz fl/fl ;Rosa26-Taz S89A fl/wt ), Yap/Taz iEC-KO ( Pdgfb-creERT2;Yap fl/fl ;Taz fl/fl ;Rosa26-Taz S89A wt/wt ) and Yap/Taz iEC-KO ; Taz iEC-GOF ( Pdgfb-creERT2;Yap fl/fl ;Taz fl/fl ;Rosa26-Taz S89A fl/wt ) mice (EC coverage: n = 16 (Ctrl), 7 ( Yap/Taz iEC-KO ) and 8 ( Yap/Taz iEC-KO ; Taz iEC-GOF ) independent samples). Western blot data in b are from the respective experiment, processed in parallel, and are representative of at least three independent experiments. For c, g , data represent mean ± s.e.m.; two-tailed unpaired t-test. * P < 0.05; ** P < 0.01; **** P < 0.0001. The numerical data, unprocessed western blots and P values are provided as source data.

Journal: Nature Metabolism

Article Title: A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth

doi: 10.1038/s42255-022-00584-y

Figure Lengend Snippet: a , Schematic representations of the wild-type Rosa26 locus, the targeting vector, the recombined as well as the excised allele are shown. A cassette containing the CAG promoter, a floxed STOP sequence, a cDNA encoding for 3xFLAG-tagged TAZ S89A and IRES-nGFP was inserted into the Rosa26 locus. Triangles denote loxP sites. DTA, diphteria toxin negative selection marker; pA, polyA signal. b , Immunoblot analysis of total lung lysates from control (Ctrl, Rosa26-Taz S89A fl/fl ) and mutant mice ( Taz iEC-GOF , Pdgfb-creERT2;Rosa26-Taz S89A fl/fl ). 4OHT was administered from P1 to P4 and samples analyzed at P6. Arrow heads indicate expression of the FLAG-tagged Taz S89A mutant. c , RT-qPCR analysis of retinal ECs at P6, showing increased expression of the canonical YAP/TAZ target gene Ctgf in Taz iEC-GOF mice when compared to Ctrl (n = 4 independent samples). d , Immunofluorescence staining for GFP, ERG and PECAM in P6 retinas depicting a nuclear GFP signal in PECAM positive vessels of Taz iEC-GOF mutants, which is absent in Ctrl mice. e , Higher magnification images of P6 retinas labelled for EdU, ERG and PECAM showing increased EC proliferation in the Taz iEC-GOF mice. f , g , Confocal images ( f ) and quantification of endothelial coverage ( g ) in PECAM labelled P6 retinas obtained from Ctrl ( Yap fl/fl ;Taz fl/fl ;Rosa26-Taz S89A fl/wt ), Yap/Taz iEC-KO ( Pdgfb-creERT2;Yap fl/fl ;Taz fl/fl ;Rosa26-Taz S89A wt/wt ) and Yap/Taz iEC-KO ; Taz iEC-GOF ( Pdgfb-creERT2;Yap fl/fl ;Taz fl/fl ;Rosa26-Taz S89A fl/wt ) mice (EC coverage: n = 16 (Ctrl), 7 ( Yap/Taz iEC-KO ) and 8 ( Yap/Taz iEC-KO ; Taz iEC-GOF ) independent samples). Western blot data in b are from the respective experiment, processed in parallel, and are representative of at least three independent experiments. For c, g , data represent mean ± s.e.m.; two-tailed unpaired t-test. * P < 0.05; ** P < 0.01; **** P < 0.0001. The numerical data, unprocessed western blots and P values are provided as source data.

Article Snippet: Human FLAG-YAP S127A (Addgene, no. 27370) and human FLAG-TAZ S89A (Addgene, no. 24815) , were subcloned into the pLVX-TetOne-Puro vector (Clontech, no. 631847).

Techniques: Plasmid Preparation, Sequencing, Selection, Marker, Western Blot, Mutagenesis, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Two Tailed Test

Journal: Nature Metabolism

Article Title: A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth

doi: 10.1038/s42255-022-00584-y

Figure Lengend Snippet: a , b , Volcano plots of proteins interacting with FLAG-tagged TAZ S89A ( a ) or YAP S127A ( b ) in HUVECs ( n = 3 independent samples). Red dots denote proteins that are significantly enriched in the TAZ S89A or YAP S127A interactome (log 2 fold change (FC) ≥ 1 and false discovery rate (FDR) < 0.05). c , d , Immunoblot analysis of endothelial TAZ ( c ) and YAP ( d ) immunoprecipitates validating the interaction of endogenous YAP/TAZ with TEADs. e , mRNA expression profile of Tead1–4 in murine ECs isolated from P6 mouse retinas as determined by RNA-seq analysis ( n = 4 independent samples). f , Transcript abundance of TEAD1–4 in HUVECs as assessed by RNA-seq ( n = 3 independent samples). g , PECAM-immunofluorescence labelling of P6 retinas illustrating a sparse vascular network in mice lacking expression of Tead1 , Tead2 and Tead4 in ECs ( Pdgfb-CreERT2;Tead1 fl/fl ;Tead2 −/− ;Tead4 fl/fl ). h , Reduced endothelial proliferation in Tead1/2/4 iEC-KO mutants as shown by EdU, ERG and PECAM colabelling of P6 retinas. Scale bars in g , h , 200 μm. i , Quantification of vascular parameters in Ctrl and Tead1/2/4 iEC-KO mice (EC area, n = 8 (Ctrl) and 6 ( Tead1/2/4 iEC-KO ) independent samples; EC number/field, n = 6 (Ctrl) and 5 ( Tead1/2/4 iEC-KO ) independent samples; EC proliferation, n = 7 (Ctrl) and 5 ( Tead1/2/4 iEC-KO ) independent samples). Western blot data in c and d are from the respective experiment, processed in parallel and are representative of at least three independent experiments. For e , f and i , data represent mean ± s.e.m.; two-tailed unpaired t -test. *** P < 0.001. The numerical data, unprocessed western blots and P values are provided as source data.

Article Snippet: Human FLAG-YAP S127A (Addgene, no. 27370) and human FLAG-TAZ S89A (Addgene, no. 24815) , were subcloned into the pLVX-TetOne-Puro vector (Clontech, no. 631847).

Techniques: Western Blot, Expressing, Isolation, RNA Sequencing Assay, Immunofluorescence, Two Tailed Test

a , Immunoblots of HUVECs transduced with doxycycline (Dox)-inducible control (Ctrl), YAP S127A (iYAP S127A ) and TAZ S89A (iTAZ S89A ) encoding lentiviruses, showing expression of FLAG-tagged YAP S127A and TAZ S89A upon Dox treatment. Samples were analyzed 48 h after treatment with Dox or vehicle. b , RT-qPCR analysis of the canonical YAP/TAZ target genes ANKRD1, CTGF, and CYR61 in iYAP S127A and iTAZ S89A expressing HUVECs. Expression changes are shown relative to Ctrl (n = 8 independent samples). c , d , Gene set enrichment analysis (GSEA) showing an enrichment of the YAP-conserved target gene expression signature in the transcriptomes of iYAP S127A ( c ) and iTAZ S89A ( d ) expressing HUVECs. ES, enrichment score; NES, normalized enrichment score. e , f , FLAG immunoprecipitation studies in HUVECs overexpressing FLAG-tagged iYAP S127A and iYAP S94A/S127A ( e ) or iTAZ S89A and iTAZ S51A/S89A ( f ). The mutation of serine 94 to alanine in YAP and serine 51 to alanine in TAZ disrupts the interaction of (nuclear) YAP and TAZ with TEADs. g , Heatmap of mRNA expression changes in HUVECs expressing Ctrl, iYAP S127A , iYAP S94A/S127A , iTAZ S89A or iTAZ S51A/S89A as determined by RNA-seq. Canonical YAP/TAZ target genes are shown (n = 3 independent samples). Western blot data in a , e and f are from the respective experiment, processed in parallel, and are representative of at least three independent experiments. For b , data represent mean ± s.e.m.; two-tailed unpaired t-test. For c , d , Kolmogorov-Smirnov test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. The numerical data, unprocessed western blots and P values are provided as source data.

Journal: Nature Metabolism

Article Title: A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth

doi: 10.1038/s42255-022-00584-y

Figure Lengend Snippet: a , Immunoblots of HUVECs transduced with doxycycline (Dox)-inducible control (Ctrl), YAP S127A (iYAP S127A ) and TAZ S89A (iTAZ S89A ) encoding lentiviruses, showing expression of FLAG-tagged YAP S127A and TAZ S89A upon Dox treatment. Samples were analyzed 48 h after treatment with Dox or vehicle. b , RT-qPCR analysis of the canonical YAP/TAZ target genes ANKRD1, CTGF, and CYR61 in iYAP S127A and iTAZ S89A expressing HUVECs. Expression changes are shown relative to Ctrl (n = 8 independent samples). c , d , Gene set enrichment analysis (GSEA) showing an enrichment of the YAP-conserved target gene expression signature in the transcriptomes of iYAP S127A ( c ) and iTAZ S89A ( d ) expressing HUVECs. ES, enrichment score; NES, normalized enrichment score. e , f , FLAG immunoprecipitation studies in HUVECs overexpressing FLAG-tagged iYAP S127A and iYAP S94A/S127A ( e ) or iTAZ S89A and iTAZ S51A/S89A ( f ). The mutation of serine 94 to alanine in YAP and serine 51 to alanine in TAZ disrupts the interaction of (nuclear) YAP and TAZ with TEADs. g , Heatmap of mRNA expression changes in HUVECs expressing Ctrl, iYAP S127A , iYAP S94A/S127A , iTAZ S89A or iTAZ S51A/S89A as determined by RNA-seq. Canonical YAP/TAZ target genes are shown (n = 3 independent samples). Western blot data in a , e and f are from the respective experiment, processed in parallel, and are representative of at least three independent experiments. For b , data represent mean ± s.e.m.; two-tailed unpaired t-test. For c , d , Kolmogorov-Smirnov test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. The numerical data, unprocessed western blots and P values are provided as source data.

Article Snippet: Human FLAG-YAP S127A (Addgene, no. 27370) and human FLAG-TAZ S89A (Addgene, no. 24815) , were subcloned into the pLVX-TetOne-Puro vector (Clontech, no. 631847).

Techniques: Western Blot, Transduction, Expressing, Quantitative RT-PCR, Immunoprecipitation, Mutagenesis, RNA Sequencing Assay, Two Tailed Test

a , b , Venn diagrams of up- ( a ) or downregulated ( b ) genes (log 2 fold change (FC) ≥1 and FDR ≤ 0.05) in HUVECs transduced with inducible YAP S127A (iYAP S127A ), TAZ S89 (iTAZ S89A ) or control (Ctrl) lentiviruses as assessed by RNA-seq. c , d , Gene set enrichment analysis plots depicting an enrichment of genes associated with activated mTORC1 signalling in HUVECs expressing iYAP S127A ( c ) or iTAZ S89A ( d ). ES, enrichment score; NES, normalized enrichment score. e , Heatmap of the enriched ‘mTORC1 signalling’ genes showing induction of these transcripts by iYAP S127A and iTAZ S89A but not by the TEAD-binding-deficient iYAP S94A/S127A iTAZ S51A/S89A mutants ( n = 3 independent samples). f , Immunoblot analysis of S6K, S6 and 4EBP1 in Ctrl, iYAP S127A and iTAZ S89A transduced HUVECs, assessing phosphorylation at mTORC1-sensitive sites. g , h , Phosphorylation status of S6K, S6 and 4EBP1 in HUVECs that were transfected with siRNAs targeting YAP / TAZ (si YAP/TAZ ) ( g ) or TEAD1 / TEAD2/TEAD4 (si TEAD1/2/4 ) ( h ). i , j , Immunolabelling of p-S6 Ser235/236 , VECAD and PECAM in P6 retinas of Ctrl, Taz iEC-GOF ( i ) and Yap/Taz iEC-KO ( j ) mutants. Scale bars, 200 μm. The isolated p-S6 Ser235/236 signal is shown in grey at the bottom. Arrows indicate the peri-venous region in Taz iEC-GOF (white) and Yap/Taz iEC-KO mice (transparent). k , Heatmap of solute carrier expression in Ctrl, iYAP S127A -, iYAP S94A/S127A -, iTAZ S89 - or iTAZ S51A/S89A -expressing HUVECs determined by RNA-seq ( n = 3 independent samples). l , m , Immunoblot analysis of SLC7A5 and SLC1A5 in si YAP/TAZ ( l ) or si TEAD1/2/4 ( m ) transfected HUVECs. n , SLC7A5 and SLC1A5 protein levels in HUVECs expressing Ctrl, iYAP S127A , iTAZ S89A , iYAP S94A/S127A or iTAZ S51A/S89A . o , TEAD-depleted HUVECs fail to induce SLC7A5 and SLC1A5 in response to iYAP S127A or ITAZ S89A overexpression as determined by immunoblotting. p , Analysis of endothelial YAP, TAZ and TEAD1 ChIP–seq peaks revealed the TEAD-binding sequence as a highly enriched motif. q , r , TAZ, YAP and TEAD1 ChIP–seq signals at the SLC7A5 ( q ) and SLC3A2 ( r ) genomic loci. RPKMs, reads per kilobase per million mapped reads. Western blot data in f – h and l – o are from the respective experiment, processed in parallel and are representative of at least three independent experiments. For c and d , the Kolmogorov–Smirnov test was used. The unprocessed blots are provided as source data.

Journal: Nature Metabolism

Article Title: A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth

doi: 10.1038/s42255-022-00584-y

Figure Lengend Snippet: a , b , Venn diagrams of up- ( a ) or downregulated ( b ) genes (log 2 fold change (FC) ≥1 and FDR ≤ 0.05) in HUVECs transduced with inducible YAP S127A (iYAP S127A ), TAZ S89 (iTAZ S89A ) or control (Ctrl) lentiviruses as assessed by RNA-seq. c , d , Gene set enrichment analysis plots depicting an enrichment of genes associated with activated mTORC1 signalling in HUVECs expressing iYAP S127A ( c ) or iTAZ S89A ( d ). ES, enrichment score; NES, normalized enrichment score. e , Heatmap of the enriched ‘mTORC1 signalling’ genes showing induction of these transcripts by iYAP S127A and iTAZ S89A but not by the TEAD-binding-deficient iYAP S94A/S127A iTAZ S51A/S89A mutants ( n = 3 independent samples). f , Immunoblot analysis of S6K, S6 and 4EBP1 in Ctrl, iYAP S127A and iTAZ S89A transduced HUVECs, assessing phosphorylation at mTORC1-sensitive sites. g , h , Phosphorylation status of S6K, S6 and 4EBP1 in HUVECs that were transfected with siRNAs targeting YAP / TAZ (si YAP/TAZ ) ( g ) or TEAD1 / TEAD2/TEAD4 (si TEAD1/2/4 ) ( h ). i , j , Immunolabelling of p-S6 Ser235/236 , VECAD and PECAM in P6 retinas of Ctrl, Taz iEC-GOF ( i ) and Yap/Taz iEC-KO ( j ) mutants. Scale bars, 200 μm. The isolated p-S6 Ser235/236 signal is shown in grey at the bottom. Arrows indicate the peri-venous region in Taz iEC-GOF (white) and Yap/Taz iEC-KO mice (transparent). k , Heatmap of solute carrier expression in Ctrl, iYAP S127A -, iYAP S94A/S127A -, iTAZ S89 - or iTAZ S51A/S89A -expressing HUVECs determined by RNA-seq ( n = 3 independent samples). l , m , Immunoblot analysis of SLC7A5 and SLC1A5 in si YAP/TAZ ( l ) or si TEAD1/2/4 ( m ) transfected HUVECs. n , SLC7A5 and SLC1A5 protein levels in HUVECs expressing Ctrl, iYAP S127A , iTAZ S89A , iYAP S94A/S127A or iTAZ S51A/S89A . o , TEAD-depleted HUVECs fail to induce SLC7A5 and SLC1A5 in response to iYAP S127A or ITAZ S89A overexpression as determined by immunoblotting. p , Analysis of endothelial YAP, TAZ and TEAD1 ChIP–seq peaks revealed the TEAD-binding sequence as a highly enriched motif. q , r , TAZ, YAP and TEAD1 ChIP–seq signals at the SLC7A5 ( q ) and SLC3A2 ( r ) genomic loci. RPKMs, reads per kilobase per million mapped reads. Western blot data in f – h and l – o are from the respective experiment, processed in parallel and are representative of at least three independent experiments. For c and d , the Kolmogorov–Smirnov test was used. The unprocessed blots are provided as source data.

Article Snippet: Human FLAG-YAP S127A (Addgene, no. 27370) and human FLAG-TAZ S89A (Addgene, no. 24815) , were subcloned into the pLVX-TetOne-Puro vector (Clontech, no. 631847).

Techniques: Transduction, RNA Sequencing Assay, Expressing, Binding Assay, Western Blot, Transfection, Isolation, Over Expression, ChIP-sequencing, Sequencing

a - c , Quantification of S6K ( a ) and S6 ( b , c ) phosphorylation in iYAP S127A or iTAZ S89A expressing ECs as determined by immunoblotting (p-S6K Thr389 : n = 6 independent samples; p-S6 Ser235/236 : n = 4 independent samples; p-S6 Ser240/244 : n = 4 independent samples). d , e , DNA ( d ) and protein ( e ) synthesis in Ctrl, iYAP S127A and iTAZ S89A HUVECs. Parameters were determined by assessing the incorporation of radiolabeled 14 C-D-glucose into DNA or 3 H-tyrosine into protein (DNA synthesis: n = 12 (Ctrl), 10 (iYAP S127A ) and 11 (iTAZ S89A ) independent samples; Protein synthesis: n = 18 (Ctrl), 16 (iYAP S127A ) and 16 (iTAZ S89A ) independent samples). f , Assessment of proliferation in iYAP S127A and iTAZ S89A expressing HUVECs as measured by 3 H-thymidine DNA incorporation (n = 15 independent samples). g , Cell counts over a 96 h period, demonstrating increased cell numbers in iYAP S127A and iTAZ S89A expressing HUVECs when compared to Ctrl (n = 9 independent samples). h - j , Quantification of S6K ( h ) and S6 ( i , j ) phosphorylation in HUVECs transfected with siRNAs targeting YAP/TAZ (si YAP/TAZ ). A pool of non-targeting siRNAs (siCtrl) was used as a control (p-S6K Thr389 : n = 8 independent samples; p-S6 Ser235/236 : n = 8 independent samples; p-S6 Ser240/244 : n = 6 independent samples). k , l , Reduction in DNA ( k ) and protein synthesis ( l ) in YAP/TAZ -depleted ECs (DNA synthesis: n = 6 (siCtrl) and 5 (si YAP/TAZ ) independent samples; Protein synthesis: n = 15 (siCtrl) and 14 (si YAP/TAZ ) independent samples). m , Reduced cell proliferation in YAP/TAZ-deficient HUVECs as assessed by 3 H-thymidine incorporation into DNA (n = 12 independent samples). n , Reduced cell counts in si YAP/TAZ versus siCtrl HUVECs (n = 9 independent samples). o-q , Quantification of S6K ( o ) and S6 ( p , q ) phosphorylation in HUVECs transfected with siRNAs targeting TEAD1 , TEAD2 and TEAD4 (si TEAD1/2/4 ). Non-targeting siRNAs (siCtrl) were used as a control (p-S6K Thr389 : n = 4 independent samples; p-S6 Ser235/236 : n = 4 independent samples; p-S6 Ser240/244 : n = 4 independent samples). For a – q , data represent mean ± s.e.m.; two-tailed unpaired t-test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. The numerical data and P values are provided as source data.

Journal: Nature Metabolism

Article Title: A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth

doi: 10.1038/s42255-022-00584-y

Figure Lengend Snippet: a - c , Quantification of S6K ( a ) and S6 ( b , c ) phosphorylation in iYAP S127A or iTAZ S89A expressing ECs as determined by immunoblotting (p-S6K Thr389 : n = 6 independent samples; p-S6 Ser235/236 : n = 4 independent samples; p-S6 Ser240/244 : n = 4 independent samples). d , e , DNA ( d ) and protein ( e ) synthesis in Ctrl, iYAP S127A and iTAZ S89A HUVECs. Parameters were determined by assessing the incorporation of radiolabeled 14 C-D-glucose into DNA or 3 H-tyrosine into protein (DNA synthesis: n = 12 (Ctrl), 10 (iYAP S127A ) and 11 (iTAZ S89A ) independent samples; Protein synthesis: n = 18 (Ctrl), 16 (iYAP S127A ) and 16 (iTAZ S89A ) independent samples). f , Assessment of proliferation in iYAP S127A and iTAZ S89A expressing HUVECs as measured by 3 H-thymidine DNA incorporation (n = 15 independent samples). g , Cell counts over a 96 h period, demonstrating increased cell numbers in iYAP S127A and iTAZ S89A expressing HUVECs when compared to Ctrl (n = 9 independent samples). h - j , Quantification of S6K ( h ) and S6 ( i , j ) phosphorylation in HUVECs transfected with siRNAs targeting YAP/TAZ (si YAP/TAZ ). A pool of non-targeting siRNAs (siCtrl) was used as a control (p-S6K Thr389 : n = 8 independent samples; p-S6 Ser235/236 : n = 8 independent samples; p-S6 Ser240/244 : n = 6 independent samples). k , l , Reduction in DNA ( k ) and protein synthesis ( l ) in YAP/TAZ -depleted ECs (DNA synthesis: n = 6 (siCtrl) and 5 (si YAP/TAZ ) independent samples; Protein synthesis: n = 15 (siCtrl) and 14 (si YAP/TAZ ) independent samples). m , Reduced cell proliferation in YAP/TAZ-deficient HUVECs as assessed by 3 H-thymidine incorporation into DNA (n = 12 independent samples). n , Reduced cell counts in si YAP/TAZ versus siCtrl HUVECs (n = 9 independent samples). o-q , Quantification of S6K ( o ) and S6 ( p , q ) phosphorylation in HUVECs transfected with siRNAs targeting TEAD1 , TEAD2 and TEAD4 (si TEAD1/2/4 ). Non-targeting siRNAs (siCtrl) were used as a control (p-S6K Thr389 : n = 4 independent samples; p-S6 Ser235/236 : n = 4 independent samples; p-S6 Ser240/244 : n = 4 independent samples). For a – q , data represent mean ± s.e.m.; two-tailed unpaired t-test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. The numerical data and P values are provided as source data.

Article Snippet: Human FLAG-YAP S127A (Addgene, no. 27370) and human FLAG-TAZ S89A (Addgene, no. 24815) , were subcloned into the pLVX-TetOne-Puro vector (Clontech, no. 631847).

Techniques: Expressing, Western Blot, DNA Synthesis, Transfection, Two Tailed Test

a , Immunolabeling of p-S6 Ser235/236 , VECAD and PECAM stained wild-type retinas showing reduced vascular growth and extinguished S6 phosphorylation in mice treated with the mTOR inhibitor rapamycin. Mice were treated with solvent (Ctrl) or rapamycin from P1-P5 and analyzed at P6. The isolated p-S6 Ser235/236 signal is shown in grey in the lower panels. A, artery; V, vein. b , Immunoblot analysis of ECs transduced with doxycycline (Dox)-inducible YAP S127A (iYAP S127A ), TAZ S89A (iTAZ S89A ) or control (Ctrl) lentiviruses, and treated with Dox as well as rapamycin or vehicle. c , Confocal images showing p-S6 Ser235/236 , VECAD and PECAM stained P6 retinas in Ctrl and Taz iEC-GOF mice after intraperitoneal administration of vehicle or rapamycin from P1 to P5. Mice were also injected with 4OHT (P1 to P4) to induce Cre -mediated recombination of the Taz GOF allele. The isolated p-S6 Ser235/236 signal is shown in grey in the lower panels. d , Analysis of amino acid consumption and release in control (siCtrl) and YAP/TAZ-deficient (si YAP/TAZ ) HUVECs as determined by LC-MS/MS (n = 3 independent samples). Western blot data in b are from the respective experiment, processed in parallel, and are representative of at least three independent experiments. For d , data represent mean ± s.e.m.; two-tailed unpaired t-test, * P < 0.05. ** P < 0.01. The numerical data, unprocessed western blots and P values are provided as source data.

Journal: Nature Metabolism

Article Title: A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth

doi: 10.1038/s42255-022-00584-y

Figure Lengend Snippet: a , Immunolabeling of p-S6 Ser235/236 , VECAD and PECAM stained wild-type retinas showing reduced vascular growth and extinguished S6 phosphorylation in mice treated with the mTOR inhibitor rapamycin. Mice were treated with solvent (Ctrl) or rapamycin from P1-P5 and analyzed at P6. The isolated p-S6 Ser235/236 signal is shown in grey in the lower panels. A, artery; V, vein. b , Immunoblot analysis of ECs transduced with doxycycline (Dox)-inducible YAP S127A (iYAP S127A ), TAZ S89A (iTAZ S89A ) or control (Ctrl) lentiviruses, and treated with Dox as well as rapamycin or vehicle. c , Confocal images showing p-S6 Ser235/236 , VECAD and PECAM stained P6 retinas in Ctrl and Taz iEC-GOF mice after intraperitoneal administration of vehicle or rapamycin from P1 to P5. Mice were also injected with 4OHT (P1 to P4) to induce Cre -mediated recombination of the Taz GOF allele. The isolated p-S6 Ser235/236 signal is shown in grey in the lower panels. d , Analysis of amino acid consumption and release in control (siCtrl) and YAP/TAZ-deficient (si YAP/TAZ ) HUVECs as determined by LC-MS/MS (n = 3 independent samples). Western blot data in b are from the respective experiment, processed in parallel, and are representative of at least three independent experiments. For d , data represent mean ± s.e.m.; two-tailed unpaired t-test, * P < 0.05. ** P < 0.01. The numerical data, unprocessed western blots and P values are provided as source data.

Article Snippet: Human FLAG-YAP S127A (Addgene, no. 27370) and human FLAG-TAZ S89A (Addgene, no. 24815) , were subcloned into the pLVX-TetOne-Puro vector (Clontech, no. 631847).

Techniques: Immunolabeling, Staining, Isolation, Western Blot, Transduction, Injection, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

a , b , ChIP-seq signals of TAZ, YAP and TEAD1 at the genomic loci of the canonical target genes ANKRD1 ( a ) and AXL ( b ). ChIP-seq signals are represented as reads per kilobase per million mapped reads (RPKMs). c , d , Slc7a5 expression in ECs isolated from the lungs of Rosa26-Taz S89A fl/fl ( c ) or Yap fl/fl ;Taz fl/fl ( d ) mice followed by transduction with control (AdCtrl) or Cre-encoding (AdCre) adenoviruses. Slc7a5 transcript levels were determined by RT-qPCR ( c , n = 6 independent samples; d , n = 6 independent samples). e , Reduced cell proliferation in SLC7A5 -deficient HUVECs (g SLC7A5 ) as measured by 3 H-thymidine DNA incorporation (n = 8 independent samples). Values are represented as fold change relative to control (gCtrl). f , Quantification of vascular parameters in Ctrl and Slc7a5 iEC-KO mice as indicated (EC area: n = 8 (Ctrl) and 10 ( Slc7a5 iEC-KO ) independent samples; EC number / field: n = 9 (Ctrl) and 11 ( Slc7a5 iEC-KO ) independent samples; EC proliferation: n = 9 (Ctrl) and 11 ( Slc7a5 iEC-KO ) independent samples). g , Immunofluorescence staining for EdU, ERG and PECAM in P6 Ctrl and Slc7a5 iEC-KO retinas. h , Confocal images of VECAD, p-S6 Ser235/236 and PECAM stained P6 retinas of Ctrl and Slc7a5 iEC-KO mice, suggesting reduced mTORC1 signaling in Slc7a5 mutants. The isolated p-S6 Ser235/236 signal is shown in grey at the bottom of the panel. i , Immunoblots of HUVECs transduced with Ctrl or SLC7A5 encoding lentivirus, showing that overexpression of SLC7A5 is insufficient to restore mTORC1 activity in YAP/TAZ -depleted HUVECs (si YAP/TAZ ). Western blot data in i are from the respective experiment, processed in parallel, and are representative of at least three independent experiments. For c , d , e - f , data represent mean ± s.e.m.; two-tailed unpaired t-test. ** P < 0.01; *** P < 0.01; **** P < 0.0001. The numerical data, unprocessed western blots and P values are provided as source data.

Journal: Nature Metabolism

Article Title: A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth

doi: 10.1038/s42255-022-00584-y

Figure Lengend Snippet: a , b , ChIP-seq signals of TAZ, YAP and TEAD1 at the genomic loci of the canonical target genes ANKRD1 ( a ) and AXL ( b ). ChIP-seq signals are represented as reads per kilobase per million mapped reads (RPKMs). c , d , Slc7a5 expression in ECs isolated from the lungs of Rosa26-Taz S89A fl/fl ( c ) or Yap fl/fl ;Taz fl/fl ( d ) mice followed by transduction with control (AdCtrl) or Cre-encoding (AdCre) adenoviruses. Slc7a5 transcript levels were determined by RT-qPCR ( c , n = 6 independent samples; d , n = 6 independent samples). e , Reduced cell proliferation in SLC7A5 -deficient HUVECs (g SLC7A5 ) as measured by 3 H-thymidine DNA incorporation (n = 8 independent samples). Values are represented as fold change relative to control (gCtrl). f , Quantification of vascular parameters in Ctrl and Slc7a5 iEC-KO mice as indicated (EC area: n = 8 (Ctrl) and 10 ( Slc7a5 iEC-KO ) independent samples; EC number / field: n = 9 (Ctrl) and 11 ( Slc7a5 iEC-KO ) independent samples; EC proliferation: n = 9 (Ctrl) and 11 ( Slc7a5 iEC-KO ) independent samples). g , Immunofluorescence staining for EdU, ERG and PECAM in P6 Ctrl and Slc7a5 iEC-KO retinas. h , Confocal images of VECAD, p-S6 Ser235/236 and PECAM stained P6 retinas of Ctrl and Slc7a5 iEC-KO mice, suggesting reduced mTORC1 signaling in Slc7a5 mutants. The isolated p-S6 Ser235/236 signal is shown in grey at the bottom of the panel. i , Immunoblots of HUVECs transduced with Ctrl or SLC7A5 encoding lentivirus, showing that overexpression of SLC7A5 is insufficient to restore mTORC1 activity in YAP/TAZ -depleted HUVECs (si YAP/TAZ ). Western blot data in i are from the respective experiment, processed in parallel, and are representative of at least three independent experiments. For c , d , e - f , data represent mean ± s.e.m.; two-tailed unpaired t-test. ** P < 0.01; *** P < 0.01; **** P < 0.0001. The numerical data, unprocessed western blots and P values are provided as source data.

Article Snippet: Human FLAG-YAP S127A (Addgene, no. 27370) and human FLAG-TAZ S89A (Addgene, no. 24815) , were subcloned into the pLVX-TetOne-Puro vector (Clontech, no. 631847).

Techniques: ChIP-sequencing, Expressing, Isolation, Transduction, Quantitative RT-PCR, Immunofluorescence, Staining, Western Blot, Over Expression, Activity Assay, Two Tailed Test

a , RNA-seq analysis of solute carrier expression in P6 mouse retinal ECs ( n = 4 independent samples). b , S6K and S6 phosphorylation in control (gCtrl) and SLC7A5-depleted (g SLC7A5 ) HUVECs. Cells were generated by CRISPR–Cas9. c , d , DNA ( c ) and protein ( d ) synthesis in gCtrl and g SLC7A5 ECs (DNA synthesis, n = 6 independent samples; protein synthesis: n = 12 independent samples; incorp., incorporation). e , Cell numbers in gCtrl and g SLC7A5 HUVECs ( n = 6 independent samples). f , g , PECAM ( f ) or ERG and PECAM ( g ) labelled P6 retinas of Ctrl ( Slc7a5 fl/fl ) and Slc7a5 iEC-KO ( Pdgfb-creERT2;Slc7a5 fl/fl ) mice. A, artery; V, vein. Scale bar f , 200 μm; g , 100 μm. h , i , S6K and S6 phosphorylation in Ctrl, iYAP S127A and iTAZ S89A HUVECs, in which SLC7A5 was inactivated by siRNA (si SLC7A5 ) ( h ) or the inhibitor JPH203 ( i ). j , RagA/B immunoblots in g RagA , g RagB and g RagA/B HUVECs. k , Cell numbers in gCtrl and g RagA/B HUVECs ( n = 6 independent samples). l , Analysis of mTORC1 activity markers in RagA/B-depleted HUVECs. m , Proliferation is compromised in RagA/B-deficient HUVECs ( n = 6 independent samples). n , o , Diminished anabolism in RagA/B-deficient HUVECs (DNA synthesis ( n ): n = 6 independent samples; protein synthesis ( o ): n = 9 independent samples). p , PECAM immunolabelling in P6 Ctrl ( RagA fl/fl ;RagB fl/fl ) and RagA/B iEC-KO ( Pdgfb-creERT2;RagA fl/fl ;RagB fl/fl ) mice. Scale bar, 200 μm. q , Vascular parameters in Ctrl and RagA/B iEC-KO mice (EC area, n = 8 (Ctrl) and 8 ( RagA/B iEC-KO ) independent samples; EC number/field, n = 8 (Ctrl) and 8 ( RagA/B iEC-KO ) independent samples; EC proliferation, n = 7 (Ctrl) and 7 ( RagA/B iEC-KO ) independent samples). r , p-S6 Ser235/236 , VECAD and PECAM labelling of P6 retinas from Ctrl and RagA/B iEC-KO mice. s , Images of P6 Ctrl and RagA/B iEC-KO retinas labelled for EdU, ERG and PECAM. Scale bars in r , s , 200 μm. t , Proliferation in Ctrl, iYAP S127A and iTAZ S89A -ECs subjected to simultaneous depletion of RagA/B ( n = 8 independent samples). u , Images of EdU, ERG and PECAM-labelled P6 retinas in Ctrl, Taz iEC-GOF and Taz iEC-GOF ;RagA/B iEC-KO mice. Scale bars, 200 μm. Immunoblotting data in b , h – j , l are representative of at least three independent experiments. For c – e , k , m – o , q and t , data represent mean ± s.e.m.; two-tailed unpaired t -test. ** P < 0.01; *** P < 0.001; **** P < 0.0001. Numerical data, unprocessed blots and P values are provided as source data.

Journal: Nature Metabolism

Article Title: A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth

doi: 10.1038/s42255-022-00584-y

Figure Lengend Snippet: a , RNA-seq analysis of solute carrier expression in P6 mouse retinal ECs ( n = 4 independent samples). b , S6K and S6 phosphorylation in control (gCtrl) and SLC7A5-depleted (g SLC7A5 ) HUVECs. Cells were generated by CRISPR–Cas9. c , d , DNA ( c ) and protein ( d ) synthesis in gCtrl and g SLC7A5 ECs (DNA synthesis, n = 6 independent samples; protein synthesis: n = 12 independent samples; incorp., incorporation). e , Cell numbers in gCtrl and g SLC7A5 HUVECs ( n = 6 independent samples). f , g , PECAM ( f ) or ERG and PECAM ( g ) labelled P6 retinas of Ctrl ( Slc7a5 fl/fl ) and Slc7a5 iEC-KO ( Pdgfb-creERT2;Slc7a5 fl/fl ) mice. A, artery; V, vein. Scale bar f , 200 μm; g , 100 μm. h , i , S6K and S6 phosphorylation in Ctrl, iYAP S127A and iTAZ S89A HUVECs, in which SLC7A5 was inactivated by siRNA (si SLC7A5 ) ( h ) or the inhibitor JPH203 ( i ). j , RagA/B immunoblots in g RagA , g RagB and g RagA/B HUVECs. k , Cell numbers in gCtrl and g RagA/B HUVECs ( n = 6 independent samples). l , Analysis of mTORC1 activity markers in RagA/B-depleted HUVECs. m , Proliferation is compromised in RagA/B-deficient HUVECs ( n = 6 independent samples). n , o , Diminished anabolism in RagA/B-deficient HUVECs (DNA synthesis ( n ): n = 6 independent samples; protein synthesis ( o ): n = 9 independent samples). p , PECAM immunolabelling in P6 Ctrl ( RagA fl/fl ;RagB fl/fl ) and RagA/B iEC-KO ( Pdgfb-creERT2;RagA fl/fl ;RagB fl/fl ) mice. Scale bar, 200 μm. q , Vascular parameters in Ctrl and RagA/B iEC-KO mice (EC area, n = 8 (Ctrl) and 8 ( RagA/B iEC-KO ) independent samples; EC number/field, n = 8 (Ctrl) and 8 ( RagA/B iEC-KO ) independent samples; EC proliferation, n = 7 (Ctrl) and 7 ( RagA/B iEC-KO ) independent samples). r , p-S6 Ser235/236 , VECAD and PECAM labelling of P6 retinas from Ctrl and RagA/B iEC-KO mice. s , Images of P6 Ctrl and RagA/B iEC-KO retinas labelled for EdU, ERG and PECAM. Scale bars in r , s , 200 μm. t , Proliferation in Ctrl, iYAP S127A and iTAZ S89A -ECs subjected to simultaneous depletion of RagA/B ( n = 8 independent samples). u , Images of EdU, ERG and PECAM-labelled P6 retinas in Ctrl, Taz iEC-GOF and Taz iEC-GOF ;RagA/B iEC-KO mice. Scale bars, 200 μm. Immunoblotting data in b , h – j , l are representative of at least three independent experiments. For c – e , k , m – o , q and t , data represent mean ± s.e.m.; two-tailed unpaired t -test. ** P < 0.01; *** P < 0.001; **** P < 0.0001. Numerical data, unprocessed blots and P values are provided as source data.

Article Snippet: Human FLAG-YAP S127A (Addgene, no. 27370) and human FLAG-TAZ S89A (Addgene, no. 24815) , were subcloned into the pLVX-TetOne-Puro vector (Clontech, no. 631847).

Techniques: RNA Sequencing Assay, Expressing, Generated, CRISPR, DNA Synthesis, Western Blot, Activity Assay, Two Tailed Test

a , RT-qPCR analysis in gCtrl and g RagA/B HUVECs, showing that RagA/B deficiency does not alter MTOR transcript levels (n = 3 independent samples). b , c , Immunoblot analysis ( b ) and quantification ( c ) of mTOR protein levels in control (gCtrl) or in RagA/B -depleted (g RagA/B ) HUVECs, confirming the RT-qPCR analysis (n = 4 independent samples). d , Analysis of mTOR/LAMP2 co-localization in RagA/B-deficient ECs as assessed by immunofluorescence imaging (n = 5 independent samples). e , Immunofluorescence images of gCtrl and g RagA/B HUVECs stained for the lysosomal protein LAMP2, mTOR, phalloidin (PHAL) and DAPI, showing reduced mTOR/LAMP2 co-localization in g RagA/B ECs when compared to gCtrl. f , Higher magnification images of P6 Ctrl and RagA/B iEC-KO mutant mice labelled for p-S6 Ser235/236 , VECAD and PECAM. The images in the lower panel show the isolated p-S6 Ser235/236 signal in grey. g , Higher magnification confocal images of P6 Ctrl and RagA/B iEC-KO retinas labelled for EdU, ERG and PECAM demonstrating a reduced number of proliferating ECs in the mutants. h , i , Suppression of DNA ( h ) and protein ( i ) synthesis in Ctrl, iYAP S127A and iTAZ S89A HUVECs subjected to simultaneous depletion of RagA/B . DNA synthesis was measured by assessing the incorporation of 14 C-glucose into DNA (n = 5 independent samples). Protein synthesis was measured by assessing the incorporation of 3 H-tyrosine into protein (n = 9 independent samples). Western blot data in b are from the respective experiment, processed in parallel, and are representative of three independent experiments. For a , c , d , h and i , data represent mean ± s.e.m.; two-tailed unpaired t-test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significant. The numerical data, unprocessed western blots and P values are provided as source data.

Journal: Nature Metabolism

Article Title: A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth

doi: 10.1038/s42255-022-00584-y

Figure Lengend Snippet: a , RT-qPCR analysis in gCtrl and g RagA/B HUVECs, showing that RagA/B deficiency does not alter MTOR transcript levels (n = 3 independent samples). b , c , Immunoblot analysis ( b ) and quantification ( c ) of mTOR protein levels in control (gCtrl) or in RagA/B -depleted (g RagA/B ) HUVECs, confirming the RT-qPCR analysis (n = 4 independent samples). d , Analysis of mTOR/LAMP2 co-localization in RagA/B-deficient ECs as assessed by immunofluorescence imaging (n = 5 independent samples). e , Immunofluorescence images of gCtrl and g RagA/B HUVECs stained for the lysosomal protein LAMP2, mTOR, phalloidin (PHAL) and DAPI, showing reduced mTOR/LAMP2 co-localization in g RagA/B ECs when compared to gCtrl. f , Higher magnification images of P6 Ctrl and RagA/B iEC-KO mutant mice labelled for p-S6 Ser235/236 , VECAD and PECAM. The images in the lower panel show the isolated p-S6 Ser235/236 signal in grey. g , Higher magnification confocal images of P6 Ctrl and RagA/B iEC-KO retinas labelled for EdU, ERG and PECAM demonstrating a reduced number of proliferating ECs in the mutants. h , i , Suppression of DNA ( h ) and protein ( i ) synthesis in Ctrl, iYAP S127A and iTAZ S89A HUVECs subjected to simultaneous depletion of RagA/B . DNA synthesis was measured by assessing the incorporation of 14 C-glucose into DNA (n = 5 independent samples). Protein synthesis was measured by assessing the incorporation of 3 H-tyrosine into protein (n = 9 independent samples). Western blot data in b are from the respective experiment, processed in parallel, and are representative of three independent experiments. For a , c , d , h and i , data represent mean ± s.e.m.; two-tailed unpaired t-test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significant. The numerical data, unprocessed western blots and P values are provided as source data.

Article Snippet: Human FLAG-YAP S127A (Addgene, no. 27370) and human FLAG-TAZ S89A (Addgene, no. 24815) , were subcloned into the pLVX-TetOne-Puro vector (Clontech, no. 631847).

Techniques: Quantitative RT-PCR, Western Blot, Immunofluorescence, Imaging, Staining, Mutagenesis, Isolation, DNA Synthesis, Two Tailed Test

A) AGS-Akata cells were transfected with constitutively active YAP (YAP(5SA)), or TAZ (TAZ(S89A)) expression vectors, or a vector control. Two days after transfection, the cells were harvested for a western blot and the expression levels of YAP, TAZ, Z, R, BMRF1, and actin (loading control) was examined. B) AGS-Akata cells were transfected with constitutively active YAP(5SA), TAZ(S89A), or a vector control. Three days after transfection the cells were harvested for an immunoblot where the expression of YAP, TAZ, Z, BMRF1, VCA-p18 and tubulin was determined. C) NOKs-Akata cells were transfected with YAP(5SA) or TAZ(S89A) expression vectors, or a vector control, and immunoblot was performed to detect expression of transfected proteins YAP and TAZ, and Z, R, BMRF1, and tubulin (loading control). D) SNU-719 gastric carcinoma cells were transfected with either YAP(5SA), TAZ(S89A), or control expression vectors. Two days after transfection the cells were harvested for a western blot, and the expression of YAP, TAZ, Z, R, BMRF1, and tubulin was assessed. Results were quantitated using ImageJ software and normalized to the loading control result for each condition. The vector control or YAP(5SA) result (if vector control had no signal) was set as 1.

Journal: PLoS Pathogens

Article Title: Hippo signaling effectors YAP and TAZ induce Epstein-Barr Virus (EBV) lytic reactivation through TEADs in epithelial cells

doi: 10.1371/journal.ppat.1009783

Figure Lengend Snippet: A) AGS-Akata cells were transfected with constitutively active YAP (YAP(5SA)), or TAZ (TAZ(S89A)) expression vectors, or a vector control. Two days after transfection, the cells were harvested for a western blot and the expression levels of YAP, TAZ, Z, R, BMRF1, and actin (loading control) was examined. B) AGS-Akata cells were transfected with constitutively active YAP(5SA), TAZ(S89A), or a vector control. Three days after transfection the cells were harvested for an immunoblot where the expression of YAP, TAZ, Z, BMRF1, VCA-p18 and tubulin was determined. C) NOKs-Akata cells were transfected with YAP(5SA) or TAZ(S89A) expression vectors, or a vector control, and immunoblot was performed to detect expression of transfected proteins YAP and TAZ, and Z, R, BMRF1, and tubulin (loading control). D) SNU-719 gastric carcinoma cells were transfected with either YAP(5SA), TAZ(S89A), or control expression vectors. Two days after transfection the cells were harvested for a western blot, and the expression of YAP, TAZ, Z, R, BMRF1, and tubulin was assessed. Results were quantitated using ImageJ software and normalized to the loading control result for each condition. The vector control or YAP(5SA) result (if vector control had no signal) was set as 1.

Article Snippet: The 3XFLAG pCMV5-TOPO TAZ (S89A) plasmid (Addgene #24815) was a gift from Jeff Wrana at the University of Toronto.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Software

A) EBV negative HeLa cells were transfected with luciferase promoter constructs that were driven by the intact Z promoter or R promoter (containing 346 or 1068 bp, respectively, of each promoter sequence relative to the transcriptional start site), or a negative control promoter (containing only 83 bp of Zp promoter sequence), along with either a vector control, or YAP(5SA), TAZ(S89A), or KLF4+BLIMP1 expression vectors. The luciferase activity produced by each promoter was measured 48 hours post-transfection. The average fold difference in luciferase activity in conditions transfected with control vector versus YAP, TAZ or KLF4/BLIMP1 vectors is shown, along with the standard error. Statistical analysis (two-sample t-test) showed results of luciferase activity of the Zp-346 vector transfected with YAP(5SA) or TAZ(S89A) vectors, versus the vector control, were significantly different as indicated. B) HeLa cells were transfected with luciferase promoter constructs that were driven by the early lytic BMRF1 promoter or the negative control promoter (Zp-83) along with either a vector control, or YAP(5SA) or TAZ(S89A) vectors. The average fold difference in luciferase activity in conditions transfected with control vector versus YAP or TAZ vectors is shown, along with the standard error and p value. C) The sequence of the BZLF1 (Zp) promoter located between -266 and -186 (relative to the transcriptional start site) is shown; suspected TEAD binding motifs, each containing a 5/6 match to the consensus TEAD binding site (CATTCC) are outlined in green. Arrows indicate locations of the 5’ Z promoter deletions. D) EBV negative HeLa cells were transfected with 5’ luciferase Z promoter constructs with either a vector control or, the YAP(5SA) expression vector, the TAZ(S89A) expression vector, or a BLIMP1 expression vector as indicated. The average fold difference in luciferase activity in conditions transfected with control vector versus the YAP(5SA), TAZ(S89A), or BLIMP1 expression vectors is shown, along with the standard error. Statistical analysis for YAP(5SA) and TAZ(S89A) induction of the Z promoter constructs -266, -226, -218, was done with the two-sample t-test. E) EBV negative HeLa cells were transfected with 5’ luciferase Z promoter constructs with either a vector control or a BLIMP1 expression vector as indicated.

Journal: PLoS Pathogens

Article Title: Hippo signaling effectors YAP and TAZ induce Epstein-Barr Virus (EBV) lytic reactivation through TEADs in epithelial cells

doi: 10.1371/journal.ppat.1009783

Figure Lengend Snippet: A) EBV negative HeLa cells were transfected with luciferase promoter constructs that were driven by the intact Z promoter or R promoter (containing 346 or 1068 bp, respectively, of each promoter sequence relative to the transcriptional start site), or a negative control promoter (containing only 83 bp of Zp promoter sequence), along with either a vector control, or YAP(5SA), TAZ(S89A), or KLF4+BLIMP1 expression vectors. The luciferase activity produced by each promoter was measured 48 hours post-transfection. The average fold difference in luciferase activity in conditions transfected with control vector versus YAP, TAZ or KLF4/BLIMP1 vectors is shown, along with the standard error. Statistical analysis (two-sample t-test) showed results of luciferase activity of the Zp-346 vector transfected with YAP(5SA) or TAZ(S89A) vectors, versus the vector control, were significantly different as indicated. B) HeLa cells were transfected with luciferase promoter constructs that were driven by the early lytic BMRF1 promoter or the negative control promoter (Zp-83) along with either a vector control, or YAP(5SA) or TAZ(S89A) vectors. The average fold difference in luciferase activity in conditions transfected with control vector versus YAP or TAZ vectors is shown, along with the standard error and p value. C) The sequence of the BZLF1 (Zp) promoter located between -266 and -186 (relative to the transcriptional start site) is shown; suspected TEAD binding motifs, each containing a 5/6 match to the consensus TEAD binding site (CATTCC) are outlined in green. Arrows indicate locations of the 5’ Z promoter deletions. D) EBV negative HeLa cells were transfected with 5’ luciferase Z promoter constructs with either a vector control or, the YAP(5SA) expression vector, the TAZ(S89A) expression vector, or a BLIMP1 expression vector as indicated. The average fold difference in luciferase activity in conditions transfected with control vector versus the YAP(5SA), TAZ(S89A), or BLIMP1 expression vectors is shown, along with the standard error. Statistical analysis for YAP(5SA) and TAZ(S89A) induction of the Z promoter constructs -266, -226, -218, was done with the two-sample t-test. E) EBV negative HeLa cells were transfected with 5’ luciferase Z promoter constructs with either a vector control or a BLIMP1 expression vector as indicated.

Article Snippet: The 3XFLAG pCMV5-TOPO TAZ (S89A) plasmid (Addgene #24815) was a gift from Jeff Wrana at the University of Toronto.

Techniques: Transfection, Luciferase, Construct, Sequencing, Negative Control, Plasmid Preparation, Expressing, Activity Assay, Produced, Binding Assay

HONE-Akata cells were transfected with either A) a vector control or co-transfected with a FLAG-tagged YAP(5SA) expression vector and TEAD1 vector, or B) a vector control or a FLAG-tagged TAZ(S89A) expression vector and TEAD1 vector. ChIP assays were performed 24 hours later using anti-FLAG antibody as described in the materials and methods. Binding of FLAG-tagged YAP(5SA) and TAZ(S89A) to various parts of the EBV genome, including the lytic Z, R and BMRF1 promoters and the latent Cp promoter was determined by qPCR. C) HONE-Akata cells were transfected with a vector control or FLAG-tagged YAP(5SA) expression vector and myc-tagged TEAD1 vector. ChIP assays were subsequently done 24 hours post transfection with a myc antibody as described in the materials and methods. Binding of myc-TEAD1 protein to the Z promoter, or negative control (NC) sites on the EBV genome was determined by qPCR. All experiments are representative of two independent biological replicates, and statistical significance was determined by the two-sample t-test.

Journal: PLoS Pathogens

Article Title: Hippo signaling effectors YAP and TAZ induce Epstein-Barr Virus (EBV) lytic reactivation through TEADs in epithelial cells

doi: 10.1371/journal.ppat.1009783

Figure Lengend Snippet: HONE-Akata cells were transfected with either A) a vector control or co-transfected with a FLAG-tagged YAP(5SA) expression vector and TEAD1 vector, or B) a vector control or a FLAG-tagged TAZ(S89A) expression vector and TEAD1 vector. ChIP assays were performed 24 hours later using anti-FLAG antibody as described in the materials and methods. Binding of FLAG-tagged YAP(5SA) and TAZ(S89A) to various parts of the EBV genome, including the lytic Z, R and BMRF1 promoters and the latent Cp promoter was determined by qPCR. C) HONE-Akata cells were transfected with a vector control or FLAG-tagged YAP(5SA) expression vector and myc-tagged TEAD1 vector. ChIP assays were subsequently done 24 hours post transfection with a myc antibody as described in the materials and methods. Binding of myc-TEAD1 protein to the Z promoter, or negative control (NC) sites on the EBV genome was determined by qPCR. All experiments are representative of two independent biological replicates, and statistical significance was determined by the two-sample t-test.

Article Snippet: The 3XFLAG pCMV5-TOPO TAZ (S89A) plasmid (Addgene #24815) was a gift from Jeff Wrana at the University of Toronto.

Techniques: Transfection, Plasmid Preparation, Expressing, Binding Assay, Negative Control

Akata Burkitt-lymphoma cells were transfected with YAP(5SA), TAZ(S89A), and TEAD1, alone or in combination with each other, along with a vector control. After 48 hours post-transfection the cells were harvested and immunoblot performed to detect expression of YAP, TAZ, TEADs, Z, BMRF1 and the loading control tubulin. Results were quantitated using ImageJ software and normalized to the loading control result for each condition. The result in cells transfected with YAP(5SA), TAZ(S89A), and TEAD1 was set as 1.

Journal: PLoS Pathogens

Article Title: Hippo signaling effectors YAP and TAZ induce Epstein-Barr Virus (EBV) lytic reactivation through TEADs in epithelial cells

doi: 10.1371/journal.ppat.1009783

Figure Lengend Snippet: Akata Burkitt-lymphoma cells were transfected with YAP(5SA), TAZ(S89A), and TEAD1, alone or in combination with each other, along with a vector control. After 48 hours post-transfection the cells were harvested and immunoblot performed to detect expression of YAP, TAZ, TEADs, Z, BMRF1 and the loading control tubulin. Results were quantitated using ImageJ software and normalized to the loading control result for each condition. The result in cells transfected with YAP(5SA), TAZ(S89A), and TEAD1 was set as 1.

Article Snippet: The 3XFLAG pCMV5-TOPO TAZ (S89A) plasmid (Addgene #24815) was a gift from Jeff Wrana at the University of Toronto.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Software

Overexpression of a constitutively activated tafazzin (TAZ) (TAZS89A) form is unable to trigger tumor development alone but cooperates with oncogenic β-catenin to induce hepatoblastoma (HB) development in mice. A: Scheme of the experiments conducted. B: Survival curves of TAZS89A and TAZS89A/ΔN90–β-catenin mice. C and D: Gross and microscopic images of TAZS89A and TAZS89A/ΔN90–β-catenin mouse livers. Note the normal histologic features of the TAZS89A-only injected liver (C), whereas almost the whole parenchyma of TAZS89A/ΔN90–β-catenin mice is occupied by tumor lesions (D). While TAZ nuclear immunoreactivity is diffused in the tumor lesions of TAZS89A/ΔN90–β-catenin mice (D), only few, single hepatocytes (arrows) express TAZ in the nucleus of TAZS89A-injected livers (C). E: Liver weight/body weight ratio of normal (wild-type) livers as well as TAZS89A and TAZS89A/ΔN90–β-catenin mice. Data are expressed as means ± SD (E). n = 5 mice per group. ∗∗P < 0.01 versus wild-type and TAZS89A mice. Scale bars: 100 μm [C, hematoxylin and eosin (H&E) staining, and C and D, TAZ staining)]; 500 μm (D, H&E staining). Original magnification: ×100 (C, H&E staining); ×200 (C and D, TAZ staining); ×40 (D, H&E staining). SB, sleeping beauty transposase; w.p.i., weeks post-injection.

Journal: The American Journal of Pathology

Article Title: The Hippo Effector Transcriptional Coactivator with PDZ-Binding Motif Cooperates with Oncogenic β-Catenin to Induce Hepatoblastoma Development in Mice and Humans

doi: 10.1016/j.ajpath.2020.03.011

Figure Lengend Snippet: Overexpression of a constitutively activated tafazzin (TAZ) (TAZS89A) form is unable to trigger tumor development alone but cooperates with oncogenic β-catenin to induce hepatoblastoma (HB) development in mice. A: Scheme of the experiments conducted. B: Survival curves of TAZS89A and TAZS89A/ΔN90–β-catenin mice. C and D: Gross and microscopic images of TAZS89A and TAZS89A/ΔN90–β-catenin mouse livers. Note the normal histologic features of the TAZS89A-only injected liver (C), whereas almost the whole parenchyma of TAZS89A/ΔN90–β-catenin mice is occupied by tumor lesions (D). While TAZ nuclear immunoreactivity is diffused in the tumor lesions of TAZS89A/ΔN90–β-catenin mice (D), only few, single hepatocytes (arrows) express TAZ in the nucleus of TAZS89A-injected livers (C). E: Liver weight/body weight ratio of normal (wild-type) livers as well as TAZS89A and TAZS89A/ΔN90–β-catenin mice. Data are expressed as means ± SD (E). n = 5 mice per group. ∗∗P < 0.01 versus wild-type and TAZS89A mice. Scale bars: 100 μm [C, hematoxylin and eosin (H&E) staining, and C and D, TAZ staining)]; 500 μm (D, H&E staining). Original magnification: ×100 (C, H&E staining); ×200 (C and D, TAZ staining); ×40 (D, H&E staining). SB, sleeping beauty transposase; w.p.i., weeks post-injection.

Article Snippet: 9 , 13 The constitutively active form of TAZ (TAZS89A) was purchased from Addgene (Cambridge, MA; plasmid 24815) and the TEAD-binding deficient form of TAZ (TAZS89AS51A) was site-mutated using PCR; the two genes were then cloned into the pT3-EF1α plasmid (pT3-EF1α-TAZS89AS51A) via Gateway cloning technology (InvitroGen, Carlsbad, CA).

Techniques: Over Expression, Injection, Staining

Histopathologic characterization of liver lesions developed in tafazzin (TAZ)S89A/ΔN90–β-catenin mice. A: Hematoxylin and eosin (H&E) staining of a TAZS89A/ΔN90–β-catenin mouse liver 1.5 weeks after hydrodynamic tail vein injection. Altered cells, characterized mainly by nuclei of various size, form little clusters in the liver parenchyma. B–D: These cells express the Myc-tagged β-catenin (B) and TAZ (C) proteins, indicating their origin from injected constructs, and are actively proliferating (D), as indicated by nuclear immunoreactivity for the proliferation marker Ki-67. E: By 3 weeks after injection, nodules of altered cells are appreciable throughout the liver. These nodular lesions occupy most of the liver surface by 6 weeks after injection. F: Lesions appear, composed of small cells with prominent nuclei and a high nuclear/cytoplasmic ratio, resembling human fetal and crowded fetal hepatoblastomas (HBs). G and H: At higher magnification, the small size of tumor cells (T) when compared to normal hepatocytes (N) can be appreciated. I: Intriguingly, together with the epithelial component shown in F, many tumors displayed the presence of a mesenchymal part, characterized by the presence of spindle cells and a stroma component. J: Within the spindle and stromal cells, epithelial/hepatoblast-like cells can be appreciated. Scale bars: 100 μm (A–D, G, I); 500 μm (E and F); 50 μm (H and J). Original magnification: ×400 (H and J); ×200 (A–D, G, I); ×40 (E and F).

Journal: The American Journal of Pathology

Article Title: The Hippo Effector Transcriptional Coactivator with PDZ-Binding Motif Cooperates with Oncogenic β-Catenin to Induce Hepatoblastoma Development in Mice and Humans

doi: 10.1016/j.ajpath.2020.03.011

Figure Lengend Snippet: Histopathologic characterization of liver lesions developed in tafazzin (TAZ)S89A/ΔN90–β-catenin mice. A: Hematoxylin and eosin (H&E) staining of a TAZS89A/ΔN90–β-catenin mouse liver 1.5 weeks after hydrodynamic tail vein injection. Altered cells, characterized mainly by nuclei of various size, form little clusters in the liver parenchyma. B–D: These cells express the Myc-tagged β-catenin (B) and TAZ (C) proteins, indicating their origin from injected constructs, and are actively proliferating (D), as indicated by nuclear immunoreactivity for the proliferation marker Ki-67. E: By 3 weeks after injection, nodules of altered cells are appreciable throughout the liver. These nodular lesions occupy most of the liver surface by 6 weeks after injection. F: Lesions appear, composed of small cells with prominent nuclei and a high nuclear/cytoplasmic ratio, resembling human fetal and crowded fetal hepatoblastomas (HBs). G and H: At higher magnification, the small size of tumor cells (T) when compared to normal hepatocytes (N) can be appreciated. I: Intriguingly, together with the epithelial component shown in F, many tumors displayed the presence of a mesenchymal part, characterized by the presence of spindle cells and a stroma component. J: Within the spindle and stromal cells, epithelial/hepatoblast-like cells can be appreciated. Scale bars: 100 μm (A–D, G, I); 500 μm (E and F); 50 μm (H and J). Original magnification: ×400 (H and J); ×200 (A–D, G, I); ×40 (E and F).

Techniques: Staining, Injection, Construct, Marker

Molecular characterization of liver lesions developed in TAZS89A/ΔN90–β-catenin mice by immunohistochemistry. Staining patterns of the epithelial (E) and the mesenchymal (M) component of TAZS89A/ΔN90–β-catenin induced tumors for several hepatocellular [hepatocyte nuclear factor (HNF)-4α, forkhead box protein (FOX)-A1, FOXA2, CCAAT/enhancer-binding protein (CEBP)-A, and glutamine synthetase (GLUL)], cholangiocellular [cytokeratin (CK)-7, CK19], and progenitor (CD44v6, homeobox protein NANOG markers are shown in each image. Scale bars = 100 μm. Original magnification, ×200. H&E, hematoxylin and eosin.

Journal: The American Journal of Pathology

Article Title: The Hippo Effector Transcriptional Coactivator with PDZ-Binding Motif Cooperates with Oncogenic β-Catenin to Induce Hepatoblastoma Development in Mice and Humans

doi: 10.1016/j.ajpath.2020.03.011

Figure Lengend Snippet: Molecular characterization of liver lesions developed in TAZS89A/ΔN90–β-catenin mice by immunohistochemistry. Staining patterns of the epithelial (E) and the mesenchymal (M) component of TAZS89A/ΔN90–β-catenin induced tumors for several hepatocellular [hepatocyte nuclear factor (HNF)-4α, forkhead box protein (FOX)-A1, FOXA2, CCAAT/enhancer-binding protein (CEBP)-A, and glutamine synthetase (GLUL)], cholangiocellular [cytokeratin (CK)-7, CK19], and progenitor (CD44v6, homeobox protein NANOG markers are shown in each image. Scale bars = 100 μm. Original magnification, ×200. H&E, hematoxylin and eosin.

Techniques: Immunohistochemistry, Staining, Binding Assay

Transcriptionally active tafazzin (TAZ) is required for hepatocarcinogenesis in association with oncogenic β-catenin. A: Scheme of the experiment. B: Survival curve of TAZS89A/ΔN90–β-catenin and TAZS89AS51A/ΔN90–β-catenin mice. TAZS89AS51A overexpression results in the induction of TAZ-dependent effects that are independent of its binding to transcriptional enhanced associate domain (TEAD) transcription factors. C: Impairment of TAZ binding to TEAD transcription factors by hydrodynamic transfection of the TAZS89AS51A plasmid inhibits TAZS89A/β-catenin carcinogenesis in the mouse liver. Histologically, the liver of TAZS89AS51A mice appears indistinguishable from that of wild-type, noninjected livers, and hepatocyte proliferation is extremely low (the arrow in the inset indicates a single Ki-67 positive cell). D: Liver weight/body weight ratio and proliferation index of wild-type as well as TAZS89A/ΔN90–β-catenin and TAZS89AS51A/ΔN90–β-catenin mice. Data are expressed as means ± SD (D). ∗∗P < 0.01 versus wild-type and TAZS89AS51A/ΔN90–β-catenin mice. Scale bar = 100 μm. Original magnification: ×200 (C, main image and inset). H&E, hematoxylin and eosin staining; SB, sleeping beauty transposase.

Journal: The American Journal of Pathology

Article Title: The Hippo Effector Transcriptional Coactivator with PDZ-Binding Motif Cooperates with Oncogenic β-Catenin to Induce Hepatoblastoma Development in Mice and Humans

doi: 10.1016/j.ajpath.2020.03.011

Figure Lengend Snippet: Transcriptionally active tafazzin (TAZ) is required for hepatocarcinogenesis in association with oncogenic β-catenin. A: Scheme of the experiment. B: Survival curve of TAZS89A/ΔN90–β-catenin and TAZS89AS51A/ΔN90–β-catenin mice. TAZS89AS51A overexpression results in the induction of TAZ-dependent effects that are independent of its binding to transcriptional enhanced associate domain (TEAD) transcription factors. C: Impairment of TAZ binding to TEAD transcription factors by hydrodynamic transfection of the TAZS89AS51A plasmid inhibits TAZS89A/β-catenin carcinogenesis in the mouse liver. Histologically, the liver of TAZS89AS51A mice appears indistinguishable from that of wild-type, noninjected livers, and hepatocyte proliferation is extremely low (the arrow in the inset indicates a single Ki-67 positive cell). D: Liver weight/body weight ratio and proliferation index of wild-type as well as TAZS89A/ΔN90–β-catenin and TAZS89AS51A/ΔN90–β-catenin mice. Data are expressed as means ± SD (D). ∗∗P < 0.01 versus wild-type and TAZS89AS51A/ΔN90–β-catenin mice. Scale bar = 100 μm. Original magnification: ×200 (C, main image and inset). H&E, hematoxylin and eosin staining; SB, sleeping beauty transposase.

Techniques: Over Expression, Binding Assay, Transfection, Plasmid Preparation, Staining

Inactivation of the Notch cascade abolishes the mesenchymal component of liver tumors, without impairing carcinogenesis, in TAZS89A/ΔN90–β-catenin mice. A: Study design. Briefly, wild-type mice were subjected to hydrodynamic tail vein injection of either TAZS89A/ΔN90–β-catenin/pT3 (control) or TAZS89A/ΔN90–β-catenin/dnRBP-J plasmid. Specifically, dnRBP-J is the dominant negative form of the transcriptional coactivator recombination signal binding protein for immunoglobulin κJ region, whose overexpression effectively inhibits the canonical Notch cascade. B: Survival curve of TAZS89A/ΔN90–β-catenin/pT3 (control) or TAZS89A/ΔN90–β-catenin/dnRBP-J mice. C: Liver lesions from TAZS89A/ΔN90–β-catenin mice are characterized both by an epithelial and a mesenchymal component. D: In contrast, the lesions from TAZS89A/ΔN90–β-catenin/dnRBP-J mice consist only of an epithelial component. Homogeneous immunohistochemical staining for the V5-tagged dnRBP-J construct throughout the lesions indicates the effective transfection of the dnRBP-J plasmid in the tumor cells. These cells are highly proliferative, as indicated by pronounced immunoreactivity for the Ki-67 proliferation marker. As expected, V5 immunoreactivity is completely negative in TAZS89A/ΔN90–β-catenin/pT3 mice (C, inset). E: Liver weight/body weight ratio of wild-type as well as TAZS89A/ΔN90–β-catenin/pT3 (control) or TAZS89A/ΔN90–β-catenin/dnRBP-J mice. Data are expressed as means ± SD (E). ∗∗P < 0.01 versus wild-type mice. Scale bars: 500 μm (C, top panel, and D, top row); 100 μm (C, bottom panel, and D, bottom row). Original magnification, ×40 (inset in C). H&E, hematoxylin and eosin staining; SB, sleeping beauty transposase.

Journal: The American Journal of Pathology

Article Title: The Hippo Effector Transcriptional Coactivator with PDZ-Binding Motif Cooperates with Oncogenic β-Catenin to Induce Hepatoblastoma Development in Mice and Humans

doi: 10.1016/j.ajpath.2020.03.011

Figure Lengend Snippet: Inactivation of the Notch cascade abolishes the mesenchymal component of liver tumors, without impairing carcinogenesis, in TAZS89A/ΔN90–β-catenin mice. A: Study design. Briefly, wild-type mice were subjected to hydrodynamic tail vein injection of either TAZS89A/ΔN90–β-catenin/pT3 (control) or TAZS89A/ΔN90–β-catenin/dnRBP-J plasmid. Specifically, dnRBP-J is the dominant negative form of the transcriptional coactivator recombination signal binding protein for immunoglobulin κJ region, whose overexpression effectively inhibits the canonical Notch cascade. B: Survival curve of TAZS89A/ΔN90–β-catenin/pT3 (control) or TAZS89A/ΔN90–β-catenin/dnRBP-J mice. C: Liver lesions from TAZS89A/ΔN90–β-catenin mice are characterized both by an epithelial and a mesenchymal component. D: In contrast, the lesions from TAZS89A/ΔN90–β-catenin/dnRBP-J mice consist only of an epithelial component. Homogeneous immunohistochemical staining for the V5-tagged dnRBP-J construct throughout the lesions indicates the effective transfection of the dnRBP-J plasmid in the tumor cells. These cells are highly proliferative, as indicated by pronounced immunoreactivity for the Ki-67 proliferation marker. As expected, V5 immunoreactivity is completely negative in TAZS89A/ΔN90–β-catenin/pT3 mice (C, inset). E: Liver weight/body weight ratio of wild-type as well as TAZS89A/ΔN90–β-catenin/pT3 (control) or TAZS89A/ΔN90–β-catenin/dnRBP-J mice. Data are expressed as means ± SD (E). ∗∗P < 0.01 versus wild-type mice. Scale bars: 500 μm (C, top panel, and D, top row); 100 μm (C, bottom panel, and D, bottom row). Original magnification, ×40 (inset in C). H&E, hematoxylin and eosin staining; SB, sleeping beauty transposase.

Techniques: Injection, Plasmid Preparation, Dominant Negative Mutation, Binding Assay, Over Expression, Immunohistochemical staining, Staining, Construct, Transfection, Marker

Inactivation of heat shock factor 1 (HSF1) does not impair liver carcinogenesis in TAZS89A/ΔN90–β-catenin mice. A: Study design. B: Survival curve of TAZS89A/ΔN90–β-catenin and TAZS89A/ΔN90–β-catenin/dominant negative (dn)-HSF1 mice. The dnHSF1 construct is a dominant negative version of HSF1 that suppresses HSF1 activity. C: Absence of any effect on tumor development in TAZS89A/ΔN90–β-catenin/dnHSF1 mice. Tumors from TAZS89A/ΔN90–β-catenin/dnHSF1 mice display the presence of the epithelial and mesenchymal component. Homogeneous immunohistochemical staining for the V5-tagged dnHSF1 construct throughout the lesions indicates the effective transfection of dnHSF1 in the tumor cells. D: Liver weight/body weight ratio of wild-type as well as TAZS89A/ΔN90–β-catenin and TAZS89A/ΔN90–β-catenin/dnHSF1 mice. Data are expressed as means ± SD (D). ∗∗P < 0.01 versus wild-type liver. Scale bars = 50 μm (C). Original magnification, ×400. H&E, hematoxylin and eosin staining; SB, sleeping beauty transposase; w.p.i., weeks post-injection.

Journal: The American Journal of Pathology

Article Title: The Hippo Effector Transcriptional Coactivator with PDZ-Binding Motif Cooperates with Oncogenic β-Catenin to Induce Hepatoblastoma Development in Mice and Humans

doi: 10.1016/j.ajpath.2020.03.011

Figure Lengend Snippet: Inactivation of heat shock factor 1 (HSF1) does not impair liver carcinogenesis in TAZS89A/ΔN90–β-catenin mice. A: Study design. B: Survival curve of TAZS89A/ΔN90–β-catenin and TAZS89A/ΔN90–β-catenin/dominant negative (dn)-HSF1 mice. The dnHSF1 construct is a dominant negative version of HSF1 that suppresses HSF1 activity. C: Absence of any effect on tumor development in TAZS89A/ΔN90–β-catenin/dnHSF1 mice. Tumors from TAZS89A/ΔN90–β-catenin/dnHSF1 mice display the presence of the epithelial and mesenchymal component. Homogeneous immunohistochemical staining for the V5-tagged dnHSF1 construct throughout the lesions indicates the effective transfection of dnHSF1 in the tumor cells. D: Liver weight/body weight ratio of wild-type as well as TAZS89A/ΔN90–β-catenin and TAZS89A/ΔN90–β-catenin/dnHSF1 mice. Data are expressed as means ± SD (D). ∗∗P < 0.01 versus wild-type liver. Scale bars = 50 μm (C). Original magnification, ×400. H&E, hematoxylin and eosin staining; SB, sleeping beauty transposase; w.p.i., weeks post-injection.

Techniques: Dominant Negative Mutation, Construct, Activity Assay, Immunohistochemical staining, Staining, Transfection, Injection

Deletion of yes-associated protein (Yap) does not affect liver carcinogenesis in TAZS89A/ΔN90–β-catenin mice. A: Study design. Briefly, Yapflox/flox conditional knockout mice were subjected to hydrodynamic tail vein injection of either TAZS89A/ΔN90–β-catenin/pCMV (control) or TAZS89A/ΔN90–β-catenin/Cre plasmid. B: Survival curve of TAZS89A/ΔN90–β-catenin/pCMV and TAZS89A/ΔN90–β-catenin/Cre mice. C: Western blot analysis showing the effective Cre-mediated deletion of Yap in the liver of TAZS89A/ΔN90–β-catenin/Cre mice. D: Liver lesions from TAZS89A/ΔN90–β-catenin/pCMV and TAZS89A/ΔN90–β-catenin/Cre mice are morphologically undistinguishable, both consisting of an epithelial and a mesenchymal component. As expected, TAZS89A/ΔN90–β-catenin/pCMV livers display nuclear accumulation of Yap in the lesions (top row), which is instead lost in corresponding lesions from TAZS89A/ΔN90–β-catenin/Cre mice. In the latter mice, Yap cytoplasmic immunoreactivity is preserved in the non-neoplastic liver surrounding the tumors (bottom row). E: Liver weight/body weight ratio of wild-type as well as TAZS89A/ΔN90–β-catenin/pCMV and TAZS89A/ΔN90–β-catenin/Cre mice. Data are expressed as means ± SD (E). ∗∗P < 0.01 versus wild-type liver. Scale bars = 100 μm. Original magnification, ×200. GAPDH, glyceraldehyde phosphate dehydrogenase; H&E, hematoxylin and eosin staining; SB, sleeping beauty transposase.

Journal: The American Journal of Pathology

Article Title: The Hippo Effector Transcriptional Coactivator with PDZ-Binding Motif Cooperates with Oncogenic β-Catenin to Induce Hepatoblastoma Development in Mice and Humans

doi: 10.1016/j.ajpath.2020.03.011

Figure Lengend Snippet: Deletion of yes-associated protein (Yap) does not affect liver carcinogenesis in TAZS89A/ΔN90–β-catenin mice. A: Study design. Briefly, Yapflox/flox conditional knockout mice were subjected to hydrodynamic tail vein injection of either TAZS89A/ΔN90–β-catenin/pCMV (control) or TAZS89A/ΔN90–β-catenin/Cre plasmid. B: Survival curve of TAZS89A/ΔN90–β-catenin/pCMV and TAZS89A/ΔN90–β-catenin/Cre mice. C: Western blot analysis showing the effective Cre-mediated deletion of Yap in the liver of TAZS89A/ΔN90–β-catenin/Cre mice. D: Liver lesions from TAZS89A/ΔN90–β-catenin/pCMV and TAZS89A/ΔN90–β-catenin/Cre mice are morphologically undistinguishable, both consisting of an epithelial and a mesenchymal component. As expected, TAZS89A/ΔN90–β-catenin/pCMV livers display nuclear accumulation of Yap in the lesions (top row), which is instead lost in corresponding lesions from TAZS89A/ΔN90–β-catenin/Cre mice. In the latter mice, Yap cytoplasmic immunoreactivity is preserved in the non-neoplastic liver surrounding the tumors (bottom row). E: Liver weight/body weight ratio of wild-type as well as TAZS89A/ΔN90–β-catenin/pCMV and TAZS89A/ΔN90–β-catenin/Cre mice. Data are expressed as means ± SD (E). ∗∗P < 0.01 versus wild-type liver. Scale bars = 100 μm. Original magnification, ×200. GAPDH, glyceraldehyde phosphate dehydrogenase; H&E, hematoxylin and eosin staining; SB, sleeping beauty transposase.

Techniques: Knock-Out, Injection, Plasmid Preparation, Western Blot, Staining

PC1-CTT binds to TAZ without disrupting the TAZ-14-3-3 interaction. (A) HEK293 cells were co-transfected with FLAG-TAZ or FLAG-YAP and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies. (B) HEK293 cells were co-transfected with FLAG-TAZ and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-HA sepharose, then blotted with the indicated antibodies. (C) A GST-tagged construct containing the C-terminal 91 amino acids of the PC1-CTT (p91) was produced in BL21 bacteria and purified on glutathione-sepharose 4B beads. The GST-p91 coated glutathione beads were then exposed to lysates from HEK293 cells expressing FLAG-TAZ and the resulting complexes were blotted with the indicated antibodies. (D) HEK293 cells were co-transfected with FLAG-TAZ(WT) or FLAG-TAZ(S89A) and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies.

Journal: Human Molecular Genetics

Article Title: Polycystin-1 regulates bone development through an interaction with the transcriptional coactivator TAZ

doi: 10.1093/hmg/ddy322

Figure Lengend Snippet: PC1-CTT binds to TAZ without disrupting the TAZ-14-3-3 interaction. (A) HEK293 cells were co-transfected with FLAG-TAZ or FLAG-YAP and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies. (B) HEK293 cells were co-transfected with FLAG-TAZ and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-HA sepharose, then blotted with the indicated antibodies. (C) A GST-tagged construct containing the C-terminal 91 amino acids of the PC1-CTT (p91) was produced in BL21 bacteria and purified on glutathione-sepharose 4B beads. The GST-p91 coated glutathione beads were then exposed to lysates from HEK293 cells expressing FLAG-TAZ and the resulting complexes were blotted with the indicated antibodies. (D) HEK293 cells were co-transfected with FLAG-TAZ(WT) or FLAG-TAZ(S89A) and HA-PC1-CTT. Cell lysates were subjected to immunoprecipitation using anti-FLAG sepharose, then blotted with the indicated antibodies.

Article Snippet: Constructs encoding 3xFLAG-TAZ(WT), 3xFLAG-TAZ(S89A) and 3xFLAG-YAP were purchased from addgene (ID# 24809, #24815, #19045).

Techniques: Transfection, Immunoprecipitation, Construct, Produced, Purification, Expressing

Expression of the PC1-CTT does not affect TAZ nuclear localization. HEK293 cells were transfected with FLAG-TAZ(WT) and FLAG-TAZ(S89A) alone or co-transfected with HA-PC1-CTT. The cells were fixed and stained with anti-FLAG (green) and anti-HA (red) antibodies and with DAPI (blue) to reveal the locations of nuclei.

Journal: Human Molecular Genetics

Article Title: Polycystin-1 regulates bone development through an interaction with the transcriptional coactivator TAZ

doi: 10.1093/hmg/ddy322

Figure Lengend Snippet: Expression of the PC1-CTT does not affect TAZ nuclear localization. HEK293 cells were transfected with FLAG-TAZ(WT) and FLAG-TAZ(S89A) alone or co-transfected with HA-PC1-CTT. The cells were fixed and stained with anti-FLAG (green) and anti-HA (red) antibodies and with DAPI (blue) to reveal the locations of nuclei.

Article Snippet: Constructs encoding 3xFLAG-TAZ(WT), 3xFLAG-TAZ(S89A) and 3xFLAG-YAP were purchased from addgene (ID# 24809, #24815, #19045).

Techniques: Expressing, Transfection, Staining

Loss of Pkd1 expression in zebrafish results in decreased jaw bone mineralization and tail curvature, which can be rescued with PC1-CTT or TAZ(S89A) overexpression. (A) Morpholinos corresponding to the zebrafish Pkd1a and Pkd1b PC1 or TAZ genes were injected into zebrafish embryos at the 1-2 cell stage to impair expression of the two Pkd1 genes and/or of TAZ. The embryos were subsequently injected with 300nM of mRNA encoding HA-PC1-CTT or TAZ(S89A), where indicated. At 7dpf the embryos were stained for calcified bone using the fluorescent calcein dye dissolved in deionized water, mounted in agarose and imaged. (B) Bone mineralization was quantified on Image-J using thresholded images. The data represent averages of three independent experiments (n = 40–100 embryos per condition); error bars represent standard error of the mean (SEM). (C and D) Morpholinos corresponding to the zebrafish Pkd1a and Pkd1b PC1 or TAZ genes were injected into zebrafish embryos at the 1–2 cell stage to impair expression of the two Pkd1 genes and/or of TAZ. The embryos were subsequently injected with 300nM of mRNA encoding HA-PC1-CTT or TAZ(S89A), where indicated. Images of embryos injected with Pkd1a/b morpholinos alone or followed by injected of mRNA encoding TAZ(S89A) are presented in Panel C. Tail curvature was scored and the results were binned into straight, mild, moderate and severe categories, as previously described (12). Knockdown of Pkd1a and Pkd1b produce a curly tail phenotype whose severity is partially ameliorated by expression of the PC1-CTT. Loss of TAZ expression prevents the PC1-CTT mediated rescue, but expression of TAZ(S89A) is sufficient to rescue the curly tail phenotype even in the absence of PC1-CTT expression.

Journal: Human Molecular Genetics

Article Title: Polycystin-1 regulates bone development through an interaction with the transcriptional coactivator TAZ

doi: 10.1093/hmg/ddy322

Figure Lengend Snippet: Loss of Pkd1 expression in zebrafish results in decreased jaw bone mineralization and tail curvature, which can be rescued with PC1-CTT or TAZ(S89A) overexpression. (A) Morpholinos corresponding to the zebrafish Pkd1a and Pkd1b PC1 or TAZ genes were injected into zebrafish embryos at the 1-2 cell stage to impair expression of the two Pkd1 genes and/or of TAZ. The embryos were subsequently injected with 300nM of mRNA encoding HA-PC1-CTT or TAZ(S89A), where indicated. At 7dpf the embryos were stained for calcified bone using the fluorescent calcein dye dissolved in deionized water, mounted in agarose and imaged. (B) Bone mineralization was quantified on Image-J using thresholded images. The data represent averages of three independent experiments (n = 40–100 embryos per condition); error bars represent standard error of the mean (SEM). (C and D) Morpholinos corresponding to the zebrafish Pkd1a and Pkd1b PC1 or TAZ genes were injected into zebrafish embryos at the 1–2 cell stage to impair expression of the two Pkd1 genes and/or of TAZ. The embryos were subsequently injected with 300nM of mRNA encoding HA-PC1-CTT or TAZ(S89A), where indicated. Images of embryos injected with Pkd1a/b morpholinos alone or followed by injected of mRNA encoding TAZ(S89A) are presented in Panel C. Tail curvature was scored and the results were binned into straight, mild, moderate and severe categories, as previously described (12). Knockdown of Pkd1a and Pkd1b produce a curly tail phenotype whose severity is partially ameliorated by expression of the PC1-CTT. Loss of TAZ expression prevents the PC1-CTT mediated rescue, but expression of TAZ(S89A) is sufficient to rescue the curly tail phenotype even in the absence of PC1-CTT expression.

Article Snippet: Constructs encoding 3xFLAG-TAZ(WT), 3xFLAG-TAZ(S89A) and 3xFLAG-YAP were purchased from addgene (ID# 24809, #24815, #19045).

Techniques: Expressing, Over Expression, Injection, Staining

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