human flag taz s89a  (Addgene inc)

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, Western Blot, Isolation, Expressing, Marker, Derivative Assay, Control, Immunofluorescence, 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 , 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, Control, 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, Immunofluorescence, 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 , 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, Control, Expressing, Quantitative RT-PCR, Targeted Gene Expression, Immunoprecipitation, Mutagenesis, RNA Sequencing, 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 , 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, Control, RNA Sequencing, Expressing, Binding Assay, Western Blot, Phospho-proteomics, Transfection, Isolation, Over Expression, ChIP-sequencing, Sequencing

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: Phospho-proteomics, Expressing, Western Blot, DNA Synthesis, Transfection, Control, 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 , 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, Phospho-proteomics, Solvent, Isolation, Western Blot, Transduction, Control, Injection, Liquid Chromatography with Mass Spectroscopy, 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 , 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, Control, Quantitative RT-PCR, Immunofluorescence, Staining, Western Blot, Over Expression, Activity Assay, 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 , 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, Expressing, Phospho-proteomics, Control, Generated, CRISPR, DNA Synthesis, Western Blot, Activity Assay, 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 , 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, Control, Immunofluorescence, Imaging, Staining, Mutagenesis, Isolation, DNA Synthesis, Two Tailed Test