mouse yap1 wt gene Search Results


gene exp yap1 hs00902712 g1  (Thermo Fisher)
94
Thermo Fisher gene exp yap1 hs00902712 g1
( A ) Tile scan maximum intensity projection of P21 femur with Pimonidazole (green) and DAPI (blue) staining. ( B ) Quantification of Pimonidazole staining intensity (artificial units, a.u.) in different organs. ( C, D ) Regional differences in Pimonidazole ( C ) and HIF2α ( D ) staining levels in metaphysis (mp) and diaphysis (dp). ( E ) Principal component analysis of RNA sequencing data using most variable genes across the samples. The first principal component (PC1) explains 63% of all variance; and PC2 13% of the variance between metaphyseal (mpECs) and diaphyseal/bone marrow (bmECs) endothelial cells. ( F, G ) Heat map showing differential expression of hypoxia ( F ) and <t>Yap1/Taz</t> ( G ) controlled genes in mpECs vs. bmECs. ( H ) Confocal image showing immunostaining of Yap1 and Taz ( H ) in 3-week-old wild-type femur. Arrowheads highlight expression in Emcn+ (red) ECs. Nuclei, DAPI (blue). ( I ) Immunostaining of Yap1 and Taz in the control (vehicle) and MG132 proteasome inhibitor-treated femoral metaphysis. ( J ) Nuclear localization (arrowheads) of Yap1 (green) in H2B-GFP+ EC nuclei (shown in red) in 3-week-old Cdh5-mTnG femoral metaphysis (mp) and bone marrow (bm). Higher magnification image shows strong Yap1 and Taz nuclear signals bmECs. ( K ) Mean intensity (a.u.) of Yap1 and Taz nuclear localization signals in bm and mp ECs. (n = 4; 48 cells in total; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). Figure 1—source data 1. Source data for .
Gene Exp Yap1 Hs00902712 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+yap1+wt+gene/pmc06970532-195-35-50?v=Thermo+Fisher
Average 94 stars, based on 1 article reviews
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gene exp mmp14 hs01037003 g1  (Thermo Fisher)
98
Thermo Fisher gene exp mmp14 hs01037003 g1
( A ) Tile scan maximum intensity projection of P21 femur with Pimonidazole (green) and DAPI (blue) staining. ( B ) Quantification of Pimonidazole staining intensity (artificial units, a.u.) in different organs. ( C, D ) Regional differences in Pimonidazole ( C ) and HIF2α ( D ) staining levels in metaphysis (mp) and diaphysis (dp). ( E ) Principal component analysis of RNA sequencing data using most variable genes across the samples. The first principal component (PC1) explains 63% of all variance; and PC2 13% of the variance between metaphyseal (mpECs) and diaphyseal/bone marrow (bmECs) endothelial cells. ( F, G ) Heat map showing differential expression of hypoxia ( F ) and <t>Yap1/Taz</t> ( G ) controlled genes in mpECs vs. bmECs. ( H ) Confocal image showing immunostaining of Yap1 and Taz ( H ) in 3-week-old wild-type femur. Arrowheads highlight expression in Emcn+ (red) ECs. Nuclei, DAPI (blue). ( I ) Immunostaining of Yap1 and Taz in the control (vehicle) and MG132 proteasome inhibitor-treated femoral metaphysis. ( J ) Nuclear localization (arrowheads) of Yap1 (green) in H2B-GFP+ EC nuclei (shown in red) in 3-week-old Cdh5-mTnG femoral metaphysis (mp) and bone marrow (bm). Higher magnification image shows strong Yap1 and Taz nuclear signals bmECs. ( K ) Mean intensity (a.u.) of Yap1 and Taz nuclear localization signals in bm and mp ECs. (n = 4; 48 cells in total; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). Figure 1—source data 1. Source data for .
Gene Exp Mmp14 Hs01037003 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 98 stars, based on 1 article reviews
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phospho yap1 ser397  (Cell Signaling Technology Inc)
95
Cell Signaling Technology Inc phospho yap1 ser397
CPPs may impair endothelial mechanotransduction. ( A ) Western blotting measurements of mechanosensitive transcription factors KLF2, KLF4, NRF2, <t>YAP1</t> and TAZ and phosphorylated forms of YAP1 and TAZ as compared to the expression of vimentin, CD31 and VE-cadherin in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to PBS group; ( B ) gene expression analysis of KLF2 , KLF4 , NFE2L2 , YAP1 , and WWTR1 genes in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. RT-qPCR measurements, the results are represented by a heat map. Gray and red colours mean fold change 0.51–1.99, and ≥2.00, respectively, compared to PBS group; ( C ) Western blotting measurements of mechanosensitive transcription factors Klf2, Klf4, Nrf2, Yap1, Taz and phosphorylated forms of Yap1 and Taz as compared to the expression of Cd31, Gapdh and histone H3 in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to NaCl group; ( D ) gene expression analysis of Klf2 , Klf4 , Nfe2l2 , Yap1 , and Wwtr1 genes in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). RT-qPCR measurements, the results are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.50, 0.51–1.99, and ≥2.00, respectively, compared to the NaCl group. HCAEC—human coronary artery endothelial cells, HITAEC—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPP—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, KLF—Krüppel-like factor, NRF—nuclear factor erythroid 2–related factor, YAP—Yes-associated protein, pYAP—phosphorylated YAP, TAZ—transcriptional co-activator with PDZ binding motif, pTAZ—phosphorylated TAZ, CD31—cluster of differentiation 31, VE-cadherin—vascular endothelial cadherin, NFE2L2—nuclear factor erythroid 2 like 2, WWTR—WW domain containing transcription regulator, Gapdh—glyceraldehyde 3-phosphate dehydrogenase, RT-qPCR—reverse transcription quantitative polymerase chain reaction.
Phospho Yap1 Ser397, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 95 stars, based on 1 article reviews
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gene exp yap1 mm00494240 m1  (Thermo Fisher)
93
Thermo Fisher gene exp yap1 mm00494240 m1
CPPs may impair endothelial mechanotransduction. ( A ) Western blotting measurements of mechanosensitive transcription factors KLF2, KLF4, NRF2, <t>YAP1</t> and TAZ and phosphorylated forms of YAP1 and TAZ as compared to the expression of vimentin, CD31 and VE-cadherin in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to PBS group; ( B ) gene expression analysis of KLF2 , KLF4 , NFE2L2 , YAP1 , and WWTR1 genes in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. RT-qPCR measurements, the results are represented by a heat map. Gray and red colours mean fold change 0.51–1.99, and ≥2.00, respectively, compared to PBS group; ( C ) Western blotting measurements of mechanosensitive transcription factors Klf2, Klf4, Nrf2, Yap1, Taz and phosphorylated forms of Yap1 and Taz as compared to the expression of Cd31, Gapdh and histone H3 in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to NaCl group; ( D ) gene expression analysis of Klf2 , Klf4 , Nfe2l2 , Yap1 , and Wwtr1 genes in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). RT-qPCR measurements, the results are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.50, 0.51–1.99, and ≥2.00, respectively, compared to the NaCl group. HCAEC—human coronary artery endothelial cells, HITAEC—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPP—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, KLF—Krüppel-like factor, NRF—nuclear factor erythroid 2–related factor, YAP—Yes-associated protein, pYAP—phosphorylated YAP, TAZ—transcriptional co-activator with PDZ binding motif, pTAZ—phosphorylated TAZ, CD31—cluster of differentiation 31, VE-cadherin—vascular endothelial cadherin, NFE2L2—nuclear factor erythroid 2 like 2, WWTR—WW domain containing transcription regulator, Gapdh—glyceraldehyde 3-phosphate dehydrogenase, RT-qPCR—reverse transcription quantitative polymerase chain reaction.
Gene Exp Yap1 Mm00494240 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+yap1+wt+gene/pmc06240092-287-7-30?v=Thermo+Fisher
Average 93 stars, based on 1 article reviews
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yap1  (Cell Signaling Technology Inc)
98
Cell Signaling Technology Inc yap1
CPPs may impair endothelial mechanotransduction. ( A ) Western blotting measurements of mechanosensitive transcription factors KLF2, KLF4, NRF2, <t>YAP1</t> and TAZ and phosphorylated forms of YAP1 and TAZ as compared to the expression of vimentin, CD31 and VE-cadherin in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to PBS group; ( B ) gene expression analysis of KLF2 , KLF4 , NFE2L2 , YAP1 , and WWTR1 genes in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. RT-qPCR measurements, the results are represented by a heat map. Gray and red colours mean fold change 0.51–1.99, and ≥2.00, respectively, compared to PBS group; ( C ) Western blotting measurements of mechanosensitive transcription factors Klf2, Klf4, Nrf2, Yap1, Taz and phosphorylated forms of Yap1 and Taz as compared to the expression of Cd31, Gapdh and histone H3 in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to NaCl group; ( D ) gene expression analysis of Klf2 , Klf4 , Nfe2l2 , Yap1 , and Wwtr1 genes in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). RT-qPCR measurements, the results are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.50, 0.51–1.99, and ≥2.00, respectively, compared to the NaCl group. HCAEC—human coronary artery endothelial cells, HITAEC—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPP—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, KLF—Krüppel-like factor, NRF—nuclear factor erythroid 2–related factor, YAP—Yes-associated protein, pYAP—phosphorylated YAP, TAZ—transcriptional co-activator with PDZ binding motif, pTAZ—phosphorylated TAZ, CD31—cluster of differentiation 31, VE-cadherin—vascular endothelial cadherin, NFE2L2—nuclear factor erythroid 2 like 2, WWTR—WW domain containing transcription regulator, Gapdh—glyceraldehyde 3-phosphate dehydrogenase, RT-qPCR—reverse transcription quantitative polymerase chain reaction.
Yap1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+yap1+wt+gene/pmc07699979-191-39-42?v=Cell+Signaling+Technology+Inc
Average 98 stars, based on 1 article reviews
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mouse yap1 wt gene  (OriGene)
90
OriGene mouse yap1 wt gene
CPPs may impair endothelial mechanotransduction. ( A ) Western blotting measurements of mechanosensitive transcription factors KLF2, KLF4, NRF2, <t>YAP1</t> and TAZ and phosphorylated forms of YAP1 and TAZ as compared to the expression of vimentin, CD31 and VE-cadherin in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to PBS group; ( B ) gene expression analysis of KLF2 , KLF4 , NFE2L2 , YAP1 , and WWTR1 genes in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. RT-qPCR measurements, the results are represented by a heat map. Gray and red colours mean fold change 0.51–1.99, and ≥2.00, respectively, compared to PBS group; ( C ) Western blotting measurements of mechanosensitive transcription factors Klf2, Klf4, Nrf2, Yap1, Taz and phosphorylated forms of Yap1 and Taz as compared to the expression of Cd31, Gapdh and histone H3 in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to NaCl group; ( D ) gene expression analysis of Klf2 , Klf4 , Nfe2l2 , Yap1 , and Wwtr1 genes in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). RT-qPCR measurements, the results are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.50, 0.51–1.99, and ≥2.00, respectively, compared to the NaCl group. HCAEC—human coronary artery endothelial cells, HITAEC—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPP—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, KLF—Krüppel-like factor, NRF—nuclear factor erythroid 2–related factor, YAP—Yes-associated protein, pYAP—phosphorylated YAP, TAZ—transcriptional co-activator with PDZ binding motif, pTAZ—phosphorylated TAZ, CD31—cluster of differentiation 31, VE-cadherin—vascular endothelial cadherin, NFE2L2—nuclear factor erythroid 2 like 2, WWTR—WW domain containing transcription regulator, Gapdh—glyceraldehyde 3-phosphate dehydrogenase, RT-qPCR—reverse transcription quantitative polymerase chain reaction.
Mouse Yap1 Wt Gene, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+yap1+wt+gene/bio_rxiv__2020__01__07__897199-343-1-15?v=OriGene
Average 90 stars, based on 1 article reviews
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mouse wt yap1 gene  (OriGene)
90
OriGene mouse wt yap1 gene
CPPs may impair endothelial mechanotransduction. ( A ) Western blotting measurements of mechanosensitive transcription factors KLF2, KLF4, NRF2, <t>YAP1</t> and TAZ and phosphorylated forms of YAP1 and TAZ as compared to the expression of vimentin, CD31 and VE-cadherin in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to PBS group; ( B ) gene expression analysis of KLF2 , KLF4 , NFE2L2 , YAP1 , and WWTR1 genes in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. RT-qPCR measurements, the results are represented by a heat map. Gray and red colours mean fold change 0.51–1.99, and ≥2.00, respectively, compared to PBS group; ( C ) Western blotting measurements of mechanosensitive transcription factors Klf2, Klf4, Nrf2, Yap1, Taz and phosphorylated forms of Yap1 and Taz as compared to the expression of Cd31, Gapdh and histone H3 in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to NaCl group; ( D ) gene expression analysis of Klf2 , Klf4 , Nfe2l2 , Yap1 , and Wwtr1 genes in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). RT-qPCR measurements, the results are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.50, 0.51–1.99, and ≥2.00, respectively, compared to the NaCl group. HCAEC—human coronary artery endothelial cells, HITAEC—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPP—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, KLF—Krüppel-like factor, NRF—nuclear factor erythroid 2–related factor, YAP—Yes-associated protein, pYAP—phosphorylated YAP, TAZ—transcriptional co-activator with PDZ binding motif, pTAZ—phosphorylated TAZ, CD31—cluster of differentiation 31, VE-cadherin—vascular endothelial cadherin, NFE2L2—nuclear factor erythroid 2 like 2, WWTR—WW domain containing transcription regulator, Gapdh—glyceraldehyde 3-phosphate dehydrogenase, RT-qPCR—reverse transcription quantitative polymerase chain reaction.
Mouse Wt Yap1 Gene, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+yap1+wt+gene/pm33046882-452-1-15?v=OriGene
Average 90 stars, based on 1 article reviews
mouse wt yap1 gene - by Bioz Stars, 2026-08
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yap1  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology yap1
Development of cataracts in the <t>Yap1</t> +/ − mice. ( A , B ) Images of WT eye ( A ) and the cataract eye of Yap1 +/ − mouse ( B ) at two months of age. ( C , D ) Transparency of freshly collected lenses. The lenses were placed on the grid papers and photographed by transillumination with white light. Transparency is indicated by the clarity of grid. ( E ) Percentage of the animals at each age group that developed cataracts. Note that each age group is a separate cohort of mice. ( F , G ) Histological hematoxylin and eosin staining of paraffin-embedded eye sections from WT and Yap1 +/ − cataract mice at three months old. The cortical rupture in Yap1 +/ − lens was noticeable in G . ( F1 , G1 ) High-magnification views of F and G, respectively. Scale bar : 500 µm ( A – D , F , and G ), 50 µm ( F1 and G1 ).
Yap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 1 article reviews
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gene exp yap1 mm01143263 m1  (Thermo Fisher)
95
Thermo Fisher gene exp yap1 mm01143263 m1
Development of cataracts in the <t>Yap1</t> +/ − mice. ( A , B ) Images of WT eye ( A ) and the cataract eye of Yap1 +/ − mouse ( B ) at two months of age. ( C , D ) Transparency of freshly collected lenses. The lenses were placed on the grid papers and photographed by transillumination with white light. Transparency is indicated by the clarity of grid. ( E ) Percentage of the animals at each age group that developed cataracts. Note that each age group is a separate cohort of mice. ( F , G ) Histological hematoxylin and eosin staining of paraffin-embedded eye sections from WT and Yap1 +/ − cataract mice at three months old. The cortical rupture in Yap1 +/ − lens was noticeable in G . ( F1 , G1 ) High-magnification views of F and G, respectively. Scale bar : 500 µm ( A – D , F , and G ), 50 µm ( F1 and G1 ).
Gene Exp Yap1 Mm01143263 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mouse+yap1+wt+gene/pm25581281-257-4--1?v=Thermo+Fisher
Average 95 stars, based on 1 article reviews
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runx paralog  (Addgene inc)
93
Addgene inc runx paralog
a, Schematic diagram shows different stages of T cell development. Hematopoietic stem and progenitor cells (HSPC), double negative (DN), double positive (DP), single positive (SP), and regulatory T cells (Treg). Representative flow cytometry plots (right) show cKit, CD25, and Bcl11b expression patterns in distinct DN populations. Note: “DN1” throughout refers only to cKit high DN1, also known as Early T Progenitor (ETP) cells. Flow cytometry data was obtained from artificial thymic organoid (ATO) culture on day 9. b, Runx1 and Runx3 occupancy patterns in non-promoter regions of indicated cell populations are shown as peak-centered heatmaps. <t>Runx</t> binding profiles in DN1 and DN3 were detected by C&R from 2 independent experiments (merged data shown), and others were reported using ChIP-seq , – . MK, Maturing megakaryocytes. Numbers indicate the percent of group among total Runx binding sites. c, Diagram illustrates the key marker gene expression patterns in early T-cell development with associated levels of Runx1+Runx3 protein (left). Different groups of non-promoter Runx binding sites in early T-development are defined (right). d, Runx1 and Runx3 occupancy patterns in Phase 1 and Phase 2 cells are shown together with ATAC and H3K4me2 profiles , . Stage-specific Runx binding groups were determined by C&R using DN1 (cKit hi CD25 - ) cells obtained from in vitro OP9-Dll1 culture and thymic DN3 (cKit low CD44 low CD25 + ) cells. e, Representative UCSC genome browser tracks for Runx C&R (independent replicates), and published ATAC-seq, and ChIP-seq data for H3K4me2, H3K27me3, CTCF, and SMC3 are shown , , . Chromatin states computed by ChromHMM are displayed as a colormap at the bottom. P1, P2: Phase 1, Phase 2. Representative Phase 1-preferential Runx binding sites (near Meis1 , left) and Phase 2-preferential sites (near Ets1 , right) are displayed. f, g, Motif analysis was conducted within constantly open chromatin sites possessing different developmental patterns of Runx binding. f, Runx motif frequency within a peak (left) and the best Runx motif quality (position weight matrix score) within each peak (right) are shown. The dotted horizontal line on the violin plot indicates threshold motif quality to score as possessing Runx motif. Thin vertical black lines mark 1.5x interquartile range and thick vertical black lines show interquartile range. Red bars with white circles indicate median values. Two sample Kolmogorov-Smirnov (KS) tests, comparing each to Group3 motif scores: *** p<0.001. g, Motif frequencies of PU.1, E2A, and ETS factors in each Runx binding site Group are displayed.
Runx Paralog, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


( A ) Tile scan maximum intensity projection of P21 femur with Pimonidazole (green) and DAPI (blue) staining. ( B ) Quantification of Pimonidazole staining intensity (artificial units, a.u.) in different organs. ( C, D ) Regional differences in Pimonidazole ( C ) and HIF2α ( D ) staining levels in metaphysis (mp) and diaphysis (dp). ( E ) Principal component analysis of RNA sequencing data using most variable genes across the samples. The first principal component (PC1) explains 63% of all variance; and PC2 13% of the variance between metaphyseal (mpECs) and diaphyseal/bone marrow (bmECs) endothelial cells. ( F, G ) Heat map showing differential expression of hypoxia ( F ) and Yap1/Taz ( G ) controlled genes in mpECs vs. bmECs. ( H ) Confocal image showing immunostaining of Yap1 and Taz ( H ) in 3-week-old wild-type femur. Arrowheads highlight expression in Emcn+ (red) ECs. Nuclei, DAPI (blue). ( I ) Immunostaining of Yap1 and Taz in the control (vehicle) and MG132 proteasome inhibitor-treated femoral metaphysis. ( J ) Nuclear localization (arrowheads) of Yap1 (green) in H2B-GFP+ EC nuclei (shown in red) in 3-week-old Cdh5-mTnG femoral metaphysis (mp) and bone marrow (bm). Higher magnification image shows strong Yap1 and Taz nuclear signals bmECs. ( K ) Mean intensity (a.u.) of Yap1 and Taz nuclear localization signals in bm and mp ECs. (n = 4; 48 cells in total; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). Figure 1—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A ) Tile scan maximum intensity projection of P21 femur with Pimonidazole (green) and DAPI (blue) staining. ( B ) Quantification of Pimonidazole staining intensity (artificial units, a.u.) in different organs. ( C, D ) Regional differences in Pimonidazole ( C ) and HIF2α ( D ) staining levels in metaphysis (mp) and diaphysis (dp). ( E ) Principal component analysis of RNA sequencing data using most variable genes across the samples. The first principal component (PC1) explains 63% of all variance; and PC2 13% of the variance between metaphyseal (mpECs) and diaphyseal/bone marrow (bmECs) endothelial cells. ( F, G ) Heat map showing differential expression of hypoxia ( F ) and Yap1/Taz ( G ) controlled genes in mpECs vs. bmECs. ( H ) Confocal image showing immunostaining of Yap1 and Taz ( H ) in 3-week-old wild-type femur. Arrowheads highlight expression in Emcn+ (red) ECs. Nuclei, DAPI (blue). ( I ) Immunostaining of Yap1 and Taz in the control (vehicle) and MG132 proteasome inhibitor-treated femoral metaphysis. ( J ) Nuclear localization (arrowheads) of Yap1 (green) in H2B-GFP+ EC nuclei (shown in red) in 3-week-old Cdh5-mTnG femoral metaphysis (mp) and bone marrow (bm). Higher magnification image shows strong Yap1 and Taz nuclear signals bmECs. ( K ) Mean intensity (a.u.) of Yap1 and Taz nuclear localization signals in bm and mp ECs. (n = 4; 48 cells in total; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). Figure 1—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: Staining, RNA Sequencing, Quantitative Proteomics, Immunostaining, Expressing, Control, Two Tailed Test

( A–B ) Confocal images of 3-week-old wild-type femoral sections showing Yap1 (green) ( A ), Taz (green) ( B ) immunostaining in the metaphyseal (mp) and bone marrow (bm) Emcn+ (red) vasculature. Nuclei (DAPI (blue). Arrows mark perivascular cells. Growth plate (gp) is indicated. ( C ) Immunostaining of Yap1 and Taz in control (vehicle) and MG132 proteasome inhibitor-treated femoral metaphysis and diaphysis. ( D ) Enhanced immunostaining of Lats2 and phospho-Yap1 S127 in the metaphysis (mp) relative to bone marrow (bm). ( E ) RNA-seq data showing expression of the Yap1, Taz ( Wwtr1 ) , Lats1 and Lats2 in mpECs and bmECs (n = 3; data are presented as mean ±sem). ( F ) In vitro culture of bone ECs from Cdh5-mTnG reporter mice. Yap1 and Taz immunostaining is concentrated in nuclei. Figure 1—figure supplement 3—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A–B ) Confocal images of 3-week-old wild-type femoral sections showing Yap1 (green) ( A ), Taz (green) ( B ) immunostaining in the metaphyseal (mp) and bone marrow (bm) Emcn+ (red) vasculature. Nuclei (DAPI (blue). Arrows mark perivascular cells. Growth plate (gp) is indicated. ( C ) Immunostaining of Yap1 and Taz in control (vehicle) and MG132 proteasome inhibitor-treated femoral metaphysis and diaphysis. ( D ) Enhanced immunostaining of Lats2 and phospho-Yap1 S127 in the metaphysis (mp) relative to bone marrow (bm). ( E ) RNA-seq data showing expression of the Yap1, Taz ( Wwtr1 ) , Lats1 and Lats2 in mpECs and bmECs (n = 3; data are presented as mean ±sem). ( F ) In vitro culture of bone ECs from Cdh5-mTnG reporter mice. Yap1 and Taz immunostaining is concentrated in nuclei. Figure 1—figure supplement 3—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: Immunostaining, Control, RNA Sequencing, Expressing, In Vitro

( A, B ) Representative confocal images of P21 control and Yap1/Taz iΔEC femoral Emcn+ (red) vasculature ( A ). Nuclei, DAPI (blue). Metaphysis (mp), transition zone (tz), diaphysis (dp), and growth plate (gp) are indicated. Note the increased number of Yap1/Taz iΔEC vessel buds and columns ( B ) compared to littermate control (n = 6, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( C, D ) Representative confocal image of Emcn+ (red) proliferating (EdU, green) mpECs. Nuclei, DAPI (blue) ( C ). Quantification of EdU + Emcn + ECs in Yap1/Taz iΔEC and control metaphysis ( D ), (control n = 6 and Yap1/Taz iΔEC n = 7, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( E, F ) Maximum intensity projections of Emcn hi (red) CD31 hi (green) vessels in the P21 Yap1/Taz iΔEC and control femur ( E ). Metaphyseal column length is significantly increased in Yap1/Taz iΔEC mutant compared to control femur ( F ) (n = 6 data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( G, H ) Representative confocal images of control and Yap1-KI iEC femur. Emcn+ (red) ECs and nuclei (DAPI, blue) are stained ( G ). Vessel buds and columns are reduced in the Yap1-KI iEC metaphysis relative to littermate control ( H ) (n = 5, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( I, J ) Maximum intensity projection of Emcn hi (red) CD31 hi (green) vessels in P21 Yap1-KI iEC and control femur. The vasculature of the metaphysis (mp) (arrows; dashed lines), the transition zone (tz) connecting the mp to the diaphysis (dp) and arteries (arrowheads) are reduced in Yap1 gain-of-function femur ( I ). The length of the Yap1-KI iEC Emcn hi CD31 hi vessel columns in femur is significantly reduced ( J ) (control n = 4 data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( K, L ) Representative confocal image of proliferating ECs (Emcn, red; EdU, green) in femoral metaphysis. Nuclei, DAPI (blue) ( K ). Quantification of EdU + Emcn + ECs in Yap1-KI iEC and control metaphysis ( L ) (control n = 4 data are presented as mean ±sem. P values, two-tailed unpaired t-test ). Figure 2—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A, B ) Representative confocal images of P21 control and Yap1/Taz iΔEC femoral Emcn+ (red) vasculature ( A ). Nuclei, DAPI (blue). Metaphysis (mp), transition zone (tz), diaphysis (dp), and growth plate (gp) are indicated. Note the increased number of Yap1/Taz iΔEC vessel buds and columns ( B ) compared to littermate control (n = 6, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( C, D ) Representative confocal image of Emcn+ (red) proliferating (EdU, green) mpECs. Nuclei, DAPI (blue) ( C ). Quantification of EdU + Emcn + ECs in Yap1/Taz iΔEC and control metaphysis ( D ), (control n = 6 and Yap1/Taz iΔEC n = 7, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( E, F ) Maximum intensity projections of Emcn hi (red) CD31 hi (green) vessels in the P21 Yap1/Taz iΔEC and control femur ( E ). Metaphyseal column length is significantly increased in Yap1/Taz iΔEC mutant compared to control femur ( F ) (n = 6 data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( G, H ) Representative confocal images of control and Yap1-KI iEC femur. Emcn+ (red) ECs and nuclei (DAPI, blue) are stained ( G ). Vessel buds and columns are reduced in the Yap1-KI iEC metaphysis relative to littermate control ( H ) (n = 5, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( I, J ) Maximum intensity projection of Emcn hi (red) CD31 hi (green) vessels in P21 Yap1-KI iEC and control femur. The vasculature of the metaphysis (mp) (arrows; dashed lines), the transition zone (tz) connecting the mp to the diaphysis (dp) and arteries (arrowheads) are reduced in Yap1 gain-of-function femur ( I ). The length of the Yap1-KI iEC Emcn hi CD31 hi vessel columns in femur is significantly reduced ( J ) (control n = 4 data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( K, L ) Representative confocal image of proliferating ECs (Emcn, red; EdU, green) in femoral metaphysis. Nuclei, DAPI (blue) ( K ). Quantification of EdU + Emcn + ECs in Yap1-KI iEC and control metaphysis ( L ) (control n = 4 data are presented as mean ±sem. P values, two-tailed unpaired t-test ). Figure 2—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: Control, Two Tailed Test, Mutagenesis, Staining

( A ) Scheme showing the time points of tamoxifen administration and analysis of endothelial specific ( Cdh5-Cre-ERT2 ) Yap1 and Wwtr1 double (Yap1/Taz iΔEC ) mutants. Mutants were sacrificed at P18 following tamoxifen treatment at P1-3 (red arrowheads) or at P21 after treatment at P6-8 (blue arrowheads), respectively. ( B, C ) Yap1 and Wwtr1 transcript levels are significantly decreased in freshly isolated Yap1/Taz iΔEC mutant bone ECs compared to control ( B ). Yap1 immunostaining is reduced in P21 Yap1/Taz iΔEC mutant bone vessels compared to control ( C ). (n = 4, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( D, E ) Representative confocal images of control and Yap1/Taz iΔEC femoral Emcn+ (red) vasculature at P18. Nuclei in ( D ), DAPI (blue). Metaphysis (mp), transition zone (tz), diaphysis (dp), and growth plate (gp) are indicated. Note the increased number of Yap1/Taz iΔEC vessel buds and arches ( E ) compared to littermate control (n = 6, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( F ) High magnification confocal images of the P18 metaphysis, transition zone, and diaphysis showing the changes in Yap1/Taz iΔEC vascular architecture relative to littermate control. ( G ) Maximum intensity projections showing ECs in P21 Yap1/Taz iΔEC Cdh5-mTnG femur compared to littermate control. Arteries (arrowheads) show high expression of Cdh5-mTnG reporter (red and green) and are Emcn (blue) negative. Graphs show quantitation of ECs in femur. (Control n = 4 and Yap1/Taz iΔEC n = 4; data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( H ) Arteries, which are negative for Emcn (blue) and covered by α-smooth muscle actin (αSMA)-positive cells (red), are significantly increased in Yap1/Taz iΔEC bone sections (n = 8, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( I ) Representative flow cytometry dot plots (top) and quantification (bar graphs, bottom) of bone ECs from control and Yap1/Taz iΔEC mutants at P21. Total ECs and type H (Emcn hi CD31 hi ) cells are increased in Yap1/Taz iΔEC mutants (n = 6, 7; data are presented as mean ±sem. P values, two-tailed unpaired t-test ). Figure 2—figure supplement 1—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A ) Scheme showing the time points of tamoxifen administration and analysis of endothelial specific ( Cdh5-Cre-ERT2 ) Yap1 and Wwtr1 double (Yap1/Taz iΔEC ) mutants. Mutants were sacrificed at P18 following tamoxifen treatment at P1-3 (red arrowheads) or at P21 after treatment at P6-8 (blue arrowheads), respectively. ( B, C ) Yap1 and Wwtr1 transcript levels are significantly decreased in freshly isolated Yap1/Taz iΔEC mutant bone ECs compared to control ( B ). Yap1 immunostaining is reduced in P21 Yap1/Taz iΔEC mutant bone vessels compared to control ( C ). (n = 4, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( D, E ) Representative confocal images of control and Yap1/Taz iΔEC femoral Emcn+ (red) vasculature at P18. Nuclei in ( D ), DAPI (blue). Metaphysis (mp), transition zone (tz), diaphysis (dp), and growth plate (gp) are indicated. Note the increased number of Yap1/Taz iΔEC vessel buds and arches ( E ) compared to littermate control (n = 6, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( F ) High magnification confocal images of the P18 metaphysis, transition zone, and diaphysis showing the changes in Yap1/Taz iΔEC vascular architecture relative to littermate control. ( G ) Maximum intensity projections showing ECs in P21 Yap1/Taz iΔEC Cdh5-mTnG femur compared to littermate control. Arteries (arrowheads) show high expression of Cdh5-mTnG reporter (red and green) and are Emcn (blue) negative. Graphs show quantitation of ECs in femur. (Control n = 4 and Yap1/Taz iΔEC n = 4; data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( H ) Arteries, which are negative for Emcn (blue) and covered by α-smooth muscle actin (αSMA)-positive cells (red), are significantly increased in Yap1/Taz iΔEC bone sections (n = 8, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( I ) Representative flow cytometry dot plots (top) and quantification (bar graphs, bottom) of bone ECs from control and Yap1/Taz iΔEC mutants at P21. Total ECs and type H (Emcn hi CD31 hi ) cells are increased in Yap1/Taz iΔEC mutants (n = 6, 7; data are presented as mean ±sem. P values, two-tailed unpaired t-test ). Figure 2—figure supplement 1—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: Isolation, Mutagenesis, Control, Immunostaining, Two Tailed Test, Expressing, Quantitation Assay, Flow Cytometry

( A ) Strategy for overexpression of constitutively active Yap1 in endothelial cells. A cassette containing the CAG promoter, a floxed STOP sequence, a cDNA encoding Yap1 S112A (in which serine residue 112 was mutated to alanine) followed by the polyadenylation signal from bovine growth hormone was inserted by homologous recombination into the Rosa26 locus. After interbreeding with Cdh5-CreE RT2 transgenic animals, Yap1 S112A expression was induced by tamoxifen administration at P1-3 followed by analysis at P6 (red arrowhead) for the analysis of retinas or at P21 (blue arrowhead) for bone samples. ( B ) Increased Yap1 (red) expression and nuclear localization (bottom panels) in the P6 Yap1-KI iEC retinal vasculature compared to control. ( C ) High magnification confocal images of metaphysis and diaphysis showing changes in Yap1 and Taz expression in Yap1-KI iEC ECs relative to littermate control. ( D ) Representative gating strategy for sorting of type H (Emcn high , CD31 high ) and type L (Emcn low , CD31 low ) bone EC subpopulations.

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A ) Strategy for overexpression of constitutively active Yap1 in endothelial cells. A cassette containing the CAG promoter, a floxed STOP sequence, a cDNA encoding Yap1 S112A (in which serine residue 112 was mutated to alanine) followed by the polyadenylation signal from bovine growth hormone was inserted by homologous recombination into the Rosa26 locus. After interbreeding with Cdh5-CreE RT2 transgenic animals, Yap1 S112A expression was induced by tamoxifen administration at P1-3 followed by analysis at P6 (red arrowhead) for the analysis of retinas or at P21 (blue arrowhead) for bone samples. ( B ) Increased Yap1 (red) expression and nuclear localization (bottom panels) in the P6 Yap1-KI iEC retinal vasculature compared to control. ( C ) High magnification confocal images of metaphysis and diaphysis showing changes in Yap1 and Taz expression in Yap1-KI iEC ECs relative to littermate control. ( D ) Representative gating strategy for sorting of type H (Emcn high , CD31 high ) and type L (Emcn low , CD31 low ) bone EC subpopulations.

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: Over Expression, Sequencing, Residue, Homologous Recombination, Transgenic Assay, Expressing, Control

( A ) Lats2 transcript levels are significantly decreased in freshly isolated Lats2 iΔEC mutant bone ECs compared to control. (n = 3, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( B, C ) Freshly dissected P21 Lats2 iΔEC mutant femur relative to littermate control ( B ). Femur length (mm) and weight are reduced in Lats2 iΔEC mutants ( C ). (control n = 7 and mutant n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( D, E ) Representative confocal images of P21 control and Lats2 iΔEC femur stained with Emcn+ (red) vasculature, and nuclei in DAPI (blue) ( D ). Vessel buds and columns are strongly reduced in Lats2 iΔEC mutants compared to littermate controls ( E ). (n = 6, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( F ) Lats2 iΔEC distal vessel buds and arches switch to a tip-like morphology (arrow). The Cdh5-mTnG reporter (red and green) visualizes nuclear fragmentation (arrowheads) in Lats2 iΔEC ECs but not in control. Taz immunosignal is strongly increased in Lats2 iΔEC mutants. ( G ) Yap1 (green) immunosignal is enhanced in Lats2 iΔEC metaphyseal vessels (Emcn, red). ( H ) Confocal image of control and Lats2 iΔEC femoral metaphysis stained for Emcn (red), CD31 (green), and nuclei (DAPI, blue). ( I, J ) Representative confocal image of proliferating (Emcn+, red; EdU+, green) ECs in metaphysis. Nuclei (DAPI, blue) ( I ). Quantification of EdU + Emcn + ECs in Lats2 iΔEC and control metaphysis ( J ) (control n = 4 and Lats2 iΔEC n = 4 data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( K, L ) Apoptotic Emcn+ (red) and active caspase-3+ (aCasp-3, green) ECs in metaphysis. Nuclei (DAPI, blue) ( K ). Quantification of Emcn + aCasp-3 + ECs ( L ) (control n = 4 and Lats2 iΔEC n = 4 data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( M ) Representative confocal image of Lats2 iΔEC Cdh5-mTnG femur compared to littermate control. Figure 3—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A ) Lats2 transcript levels are significantly decreased in freshly isolated Lats2 iΔEC mutant bone ECs compared to control. (n = 3, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( B, C ) Freshly dissected P21 Lats2 iΔEC mutant femur relative to littermate control ( B ). Femur length (mm) and weight are reduced in Lats2 iΔEC mutants ( C ). (control n = 7 and mutant n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( D, E ) Representative confocal images of P21 control and Lats2 iΔEC femur stained with Emcn+ (red) vasculature, and nuclei in DAPI (blue) ( D ). Vessel buds and columns are strongly reduced in Lats2 iΔEC mutants compared to littermate controls ( E ). (n = 6, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( F ) Lats2 iΔEC distal vessel buds and arches switch to a tip-like morphology (arrow). The Cdh5-mTnG reporter (red and green) visualizes nuclear fragmentation (arrowheads) in Lats2 iΔEC ECs but not in control. Taz immunosignal is strongly increased in Lats2 iΔEC mutants. ( G ) Yap1 (green) immunosignal is enhanced in Lats2 iΔEC metaphyseal vessels (Emcn, red). ( H ) Confocal image of control and Lats2 iΔEC femoral metaphysis stained for Emcn (red), CD31 (green), and nuclei (DAPI, blue). ( I, J ) Representative confocal image of proliferating (Emcn+, red; EdU+, green) ECs in metaphysis. Nuclei (DAPI, blue) ( I ). Quantification of EdU + Emcn + ECs in Lats2 iΔEC and control metaphysis ( J ) (control n = 4 and Lats2 iΔEC n = 4 data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( K, L ) Apoptotic Emcn+ (red) and active caspase-3+ (aCasp-3, green) ECs in metaphysis. Nuclei (DAPI, blue) ( K ). Quantification of Emcn + aCasp-3 + ECs ( L ) (control n = 4 and Lats2 iΔEC n = 4 data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( M ) Representative confocal image of Lats2 iΔEC Cdh5-mTnG femur compared to littermate control. Figure 3—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: Isolation, Mutagenesis, Control, Two Tailed Test, Staining

( A ) Scheme showing the time points of tamoxifen administration and analysis of Lats2 iΔEC mutants. ( B, C ) Average body weight ( B ) and tibia weight and length ( C ) of control and Lats2 iΔEC mutants (Control n = 7; mutant = 6). Data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( D ) Representative images of P21 control and Lats2 iΔEC femoral epiphysis with vessels (Emcn, red) in secondary ossification center. Nuclei, DAPI (blue). ( E ) Representative images of 3-week-old Cdh5-mTnG (red and green) control and Lats2 iΔEC mutant bones. ( F ) Representative images of P21 control and Lats2 iΔEC femoral metaphyseal vessels stained for Emcn (red) and VEGFR3 (green). VEGFR3, a marker of vessel buds in proximity of the growth plate, is reduced in Lats2 iΔEC mutants. ( G ) Graph showing the results of the flow cytometric analysis of the number of Emcn+ CD31+ ECs (n = 4, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( H, I ) Representative images of P21 Cdh5-mTnG (red and green) control and Lats2 iΔEC bones. Yap1 ( H ) and Taz ( I ) expression and tip-like cells (arrow) are increased in mutant metaphyseal vessels. ( J ) Representative images of P21 control and Lats2 iΔEC femoral increased Taz immunosignal (green) in the Lats2 iΔEC metaphyseal vasculature (Emcn, red) relative to control. ( K ) High magnification of confocal image showing that buds are replaced by sprout-like structures in Lats2 iΔEC mutants. Figure 3—figure supplement 1—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A ) Scheme showing the time points of tamoxifen administration and analysis of Lats2 iΔEC mutants. ( B, C ) Average body weight ( B ) and tibia weight and length ( C ) of control and Lats2 iΔEC mutants (Control n = 7; mutant = 6). Data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( D ) Representative images of P21 control and Lats2 iΔEC femoral epiphysis with vessels (Emcn, red) in secondary ossification center. Nuclei, DAPI (blue). ( E ) Representative images of 3-week-old Cdh5-mTnG (red and green) control and Lats2 iΔEC mutant bones. ( F ) Representative images of P21 control and Lats2 iΔEC femoral metaphyseal vessels stained for Emcn (red) and VEGFR3 (green). VEGFR3, a marker of vessel buds in proximity of the growth plate, is reduced in Lats2 iΔEC mutants. ( G ) Graph showing the results of the flow cytometric analysis of the number of Emcn+ CD31+ ECs (n = 4, data are presented as mean ±sem. P values, two-tailed unpaired t-test ). ( H, I ) Representative images of P21 Cdh5-mTnG (red and green) control and Lats2 iΔEC bones. Yap1 ( H ) and Taz ( I ) expression and tip-like cells (arrow) are increased in mutant metaphyseal vessels. ( J ) Representative images of P21 control and Lats2 iΔEC femoral increased Taz immunosignal (green) in the Lats2 iΔEC metaphyseal vasculature (Emcn, red) relative to control. ( K ) High magnification of confocal image showing that buds are replaced by sprout-like structures in Lats2 iΔEC mutants. Figure 3—figure supplement 1—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: Control, Mutagenesis, Two Tailed Test, Staining, Marker, Expressing

( A, B ) P6 retina whole-mount immunostaining of Yap1 ( A ), green) and Taz ( B ), green) shows expression of Isolectin B4-positive (IB4, red) retinal vessels. Higher magnifications show sprouts at the angiogenic front (center) and arteries ( A ) and veins ( V ) in the central plexus. ( C–E ) Confocal images of P6 retina whole-mount showing the Isolectin B4-stained Yap1/Taz iΔEC and littermate control vasculature ( C ). Higher magnification images show staining with Isolectin B4 (blue), anti-Erg antibody (EC nuclei, red) and EdU (green) cells. Nuclei of EdU+ ECs appear white ( D ). Reduction of vascular coverage, branching, tip cells, number of Erg+ ECs, and percentage of proliferating Erg+ EdU+ ECs in Yap1/Taz iΔEC mutant retinal vessels ( E ) (n = 6 and 4; data are presented as mean ±sem. P values, two-tailed unpaired t-test ). Figure 3—figure supplement 2—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A, B ) P6 retina whole-mount immunostaining of Yap1 ( A ), green) and Taz ( B ), green) shows expression of Isolectin B4-positive (IB4, red) retinal vessels. Higher magnifications show sprouts at the angiogenic front (center) and arteries ( A ) and veins ( V ) in the central plexus. ( C–E ) Confocal images of P6 retina whole-mount showing the Isolectin B4-stained Yap1/Taz iΔEC and littermate control vasculature ( C ). Higher magnification images show staining with Isolectin B4 (blue), anti-Erg antibody (EC nuclei, red) and EdU (green) cells. Nuclei of EdU+ ECs appear white ( D ). Reduction of vascular coverage, branching, tip cells, number of Erg+ ECs, and percentage of proliferating Erg+ EdU+ ECs in Yap1/Taz iΔEC mutant retinal vessels ( E ) (n = 6 and 4; data are presented as mean ±sem. P values, two-tailed unpaired t-test ). Figure 3—figure supplement 2—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: Immunostaining, Expressing, Staining, Control, Mutagenesis, Two Tailed Test

( A ) Principal component analysis (PCA) of RNA sequencing data from control and Yap1/Taz iΔEC bone ECs. ( B ) Heatmap of selected upregulated and downregulated genes in Yap1/Taz iΔEC bone ECs. ( C ) Gene ontology (GO) analysis of Yap1/Taz iΔEC upregulated genes. Size of the bar graph represents significance based on p-value. ( D ) PCA of RNA sequencing data from control and Lats2 iΔEC mutant bone ECs. ( E ) Differentially regulated genes shown in MA-plots of control and Lats2 iΔEC bone ECs. The x-axis represents the mean normalized counts and the y-axis shows the log2 fold change. ( F–H ) Heatmap of selected gene sets showing changes in molecular markers for bone marrow ECs ( F ), tip cells ( G ) and pro-apoptotic genes ( H ). Figure 4—figure supplement 1—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A ) Principal component analysis (PCA) of RNA sequencing data from control and Yap1/Taz iΔEC bone ECs. ( B ) Heatmap of selected upregulated and downregulated genes in Yap1/Taz iΔEC bone ECs. ( C ) Gene ontology (GO) analysis of Yap1/Taz iΔEC upregulated genes. Size of the bar graph represents significance based on p-value. ( D ) PCA of RNA sequencing data from control and Lats2 iΔEC mutant bone ECs. ( E ) Differentially regulated genes shown in MA-plots of control and Lats2 iΔEC bone ECs. The x-axis represents the mean normalized counts and the y-axis shows the log2 fold change. ( F–H ) Heatmap of selected gene sets showing changes in molecular markers for bone marrow ECs ( F ), tip cells ( G ) and pro-apoptotic genes ( H ). Figure 4—figure supplement 1—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: RNA Sequencing, Control, Mutagenesis

( A ) Differentially expressed genes determined by RNA-seq analysis of ECs from 3-week-old control vs. Yap1/Taz iΔEC mutant bone (n = 3; log2 fold change). ( B ) Bar graph showing enrichment of cellular signaling pathway components among Yap1/Taz iΔEC upregulated genes. ( C ) Heatmap of upregulated HIF1α target genes in Yap1/Taz iΔEC bone ECs. ( D ) RNA-seq data showing expression of the HIF1α target genes Vegfa , Lrg1, Angptl4 , and Xbp1 in control and mutant bone ECs (n = 3; data are presented as mean ±sem). ( E, F ) Heatmaps of Hippo pathway ( E ) and hypoxia ( F ) target genes. Expression of Cyr61, Thbs1, Ddah1, Lhfp, and Ctgf is increased in Lats2 iΔEC ECs relative to control, whereas the HIF1α targets Lrg1, Xbp1, Angptl4, Igfbp2 , and Vegfa are decreased. ( G ) Bar graph showing Ctgf, Cyr61, Vegfa and Angptl4 gene expression levels in control and Lats2 iΔEC mutant ECs. ( H, I ) Expression of the Yap1/Taz targets Ctgf, Cyr61 and the HIF1α targets Vegfa, Angptl4 in freshly isolated type H and type L EC subpopulations from P21 Yap1/Taz iΔEC ( H ) and Lats2 iΔEC ( I ) bone samples. Figure 4—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A ) Differentially expressed genes determined by RNA-seq analysis of ECs from 3-week-old control vs. Yap1/Taz iΔEC mutant bone (n = 3; log2 fold change). ( B ) Bar graph showing enrichment of cellular signaling pathway components among Yap1/Taz iΔEC upregulated genes. ( C ) Heatmap of upregulated HIF1α target genes in Yap1/Taz iΔEC bone ECs. ( D ) RNA-seq data showing expression of the HIF1α target genes Vegfa , Lrg1, Angptl4 , and Xbp1 in control and mutant bone ECs (n = 3; data are presented as mean ±sem). ( E, F ) Heatmaps of Hippo pathway ( E ) and hypoxia ( F ) target genes. Expression of Cyr61, Thbs1, Ddah1, Lhfp, and Ctgf is increased in Lats2 iΔEC ECs relative to control, whereas the HIF1α targets Lrg1, Xbp1, Angptl4, Igfbp2 , and Vegfa are decreased. ( G ) Bar graph showing Ctgf, Cyr61, Vegfa and Angptl4 gene expression levels in control and Lats2 iΔEC mutant ECs. ( H, I ) Expression of the Yap1/Taz targets Ctgf, Cyr61 and the HIF1α targets Vegfa, Angptl4 in freshly isolated type H and type L EC subpopulations from P21 Yap1/Taz iΔEC ( H ) and Lats2 iΔEC ( I ) bone samples. Figure 4—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: RNA Sequencing, Control, Mutagenesis, Expressing, Gene Expression, Isolation

( A ) Increased expression of the HIF1α target genes VEGFA and ANGPTL4 as well as Yap1/Taz target genes CTGF and CYR61 under hypoxic condition (1% O 2 ) compared to normoxia (21% O 2 ) (n = 4; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( B ) Confocal image of HUVEC showing nuclear accumulation of Yap1/Taz in 1% O 2 relative to 21% O 2 . Nuclei, DAPI (blue). ( C ) Increased expression of the Yap1/Taz target genes CTGF, CYR61 and the HIF1α target VEGFA in HUVECs in 1% O 2 relative to 21% O 2 . CTGF and CYR61 expression under both conditions is significantly reduced in siYAP1/TAZ -transfected (Y/T) HUVECs. VEGFA expression significantly increased in Y/T cells in both conditions (n = 4; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( D ) Western blots showing immunoprecipitation (IP) of Yap1/Taz followed by immunoblotting of HIF1α and Yap1/Taz. IgG is used as negative control in IP. Lysates are shown as loading control. ( E ) Increased VEGFA , ANGPTL4 , IGFBP2 expression in siYAP1/TAZ -treated HUVECs is normalized by siHIF1A transfection, whereas baseline VEGFA , ANGPTL4 and IGFBP2 is not altered by HIF1A knockdown alone (n = 3–4; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( F ) Enrichment of VEGFA promoter sequences after chromatin immunoprecipitation with HIF1α antibodies compared to IgG antibodies under hypoxic conditions and after transfection of HUVECs with siYAP1/TAZ or siControl (n = 3; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). Figure 5—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A ) Increased expression of the HIF1α target genes VEGFA and ANGPTL4 as well as Yap1/Taz target genes CTGF and CYR61 under hypoxic condition (1% O 2 ) compared to normoxia (21% O 2 ) (n = 4; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( B ) Confocal image of HUVEC showing nuclear accumulation of Yap1/Taz in 1% O 2 relative to 21% O 2 . Nuclei, DAPI (blue). ( C ) Increased expression of the Yap1/Taz target genes CTGF, CYR61 and the HIF1α target VEGFA in HUVECs in 1% O 2 relative to 21% O 2 . CTGF and CYR61 expression under both conditions is significantly reduced in siYAP1/TAZ -transfected (Y/T) HUVECs. VEGFA expression significantly increased in Y/T cells in both conditions (n = 4; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( D ) Western blots showing immunoprecipitation (IP) of Yap1/Taz followed by immunoblotting of HIF1α and Yap1/Taz. IgG is used as negative control in IP. Lysates are shown as loading control. ( E ) Increased VEGFA , ANGPTL4 , IGFBP2 expression in siYAP1/TAZ -treated HUVECs is normalized by siHIF1A transfection, whereas baseline VEGFA , ANGPTL4 and IGFBP2 is not altered by HIF1A knockdown alone (n = 3–4; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( F ) Enrichment of VEGFA promoter sequences after chromatin immunoprecipitation with HIF1α antibodies compared to IgG antibodies under hypoxic conditions and after transfection of HUVECs with siYAP1/TAZ or siControl (n = 3; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). Figure 5—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: Expressing, Two Tailed Test, Transfection, Western Blot, Immunoprecipitation, Negative Control, Control, Knockdown, Chromatin Immunoprecipitation

( A ) Decreased Yap1 and Taz protein levels in siYAP1 and siTAZ -transfected HUVECs. ( B ) Reduced expression of YAP1 , TAZ and HIF1A transcripts in HUVECs transfected with the indicated siRNAs. ( C ) XBP1 expression is increased in siYAP1/TAZ -treated HUVECs and normalized by siHIF1A, whereas baseline XBP1 is not altered by HIF1A knockdown alone (n = 3–4; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( D ) Expression of the Yap1/Taz targets Ctgf, Cyr61 and the HIF1α targets Vegfa, Angptl4 in freshly isolated type H and type L EC subpopulations from P21 Hif1a iΔEC littermate control long bone. Figure 5—figure supplement 1—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A ) Decreased Yap1 and Taz protein levels in siYAP1 and siTAZ -transfected HUVECs. ( B ) Reduced expression of YAP1 , TAZ and HIF1A transcripts in HUVECs transfected with the indicated siRNAs. ( C ) XBP1 expression is increased in siYAP1/TAZ -treated HUVECs and normalized by siHIF1A, whereas baseline XBP1 is not altered by HIF1A knockdown alone (n = 3–4; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( D ) Expression of the Yap1/Taz targets Ctgf, Cyr61 and the HIF1α targets Vegfa, Angptl4 in freshly isolated type H and type L EC subpopulations from P21 Hif1a iΔEC littermate control long bone. Figure 5—figure supplement 1—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: Transfection, Expressing, Knockdown, Two Tailed Test, Isolation, Control

( A, B ) Representative confocal images of control and HIF1AdPA iEC femur. Emcn+ (red) ECs and nuclei (DAPI, blue) are stained ( A ). Vessel buds and column are increased in the HIF1AdPA iEC metaphysis relative to littermate control ( B ) (n = 5; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( C ) Metaphyseal Emcn hi (red) CD31 hi (green) capillaries in P21 control and HIF1A gain-of-function (HIF1AdPA iEC ) femur. Nuclei, DAPI (blue). Number of aSMA positive arteries is increased in HIF1AdPA iEC femur compared to control. ( D, E ) Maximum intensity projections of Yap1/Taz iΔEC , Yap1/Taz; Hif1a iΔEC and control femur stained for Emcn (red) and CD31 (green). Nuclei, DAPI (blue). Arrowheads mark vessels in transition zone ( D ). Quantitative analysis of length and number of vessel column ( E ) (n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). Figure 6—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A, B ) Representative confocal images of control and HIF1AdPA iEC femur. Emcn+ (red) ECs and nuclei (DAPI, blue) are stained ( A ). Vessel buds and column are increased in the HIF1AdPA iEC metaphysis relative to littermate control ( B ) (n = 5; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( C ) Metaphyseal Emcn hi (red) CD31 hi (green) capillaries in P21 control and HIF1A gain-of-function (HIF1AdPA iEC ) femur. Nuclei, DAPI (blue). Number of aSMA positive arteries is increased in HIF1AdPA iEC femur compared to control. ( D, E ) Maximum intensity projections of Yap1/Taz iΔEC , Yap1/Taz; Hif1a iΔEC and control femur stained for Emcn (red) and CD31 (green). Nuclei, DAPI (blue). Arrowheads mark vessels in transition zone ( D ). Quantitative analysis of length and number of vessel column ( E ) (n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). Figure 6—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: Control, Staining, Two Tailed Test

( A ) Average body weight of P21 control and Yap1/Taz iΔEC mutants (n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( B, C ) Representative images of P21 control and Yap1/Taz iΔEC femur ( C ). Quantitation of femur length and weight show no changes between Yap1/Taz iΔEC mutants and littermate controls ( C ) (n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( D ) Confocal images showing Osterix+ cells (green) in relation to Emcn+ ECs (red) in P21 Yap1/Taz iΔEC and control metaphysis. Graph on the right shows quantitative analysis of Osterix+ (Osx+) cells (control n = 6 and Yap1/Taz iΔEC n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( E ) Confocal images showing bone vessels (Emcn), and the bone matrix protein Osteopontin in Yap1/Taz iΔEC vs control femur. ( F ) Representative confocal images of Osterix+ cells (green) in relation to Emcn+ ECs (red) in the P21 control and Yap1-KI iEC femoral metaphysis. Graph on the right shows significant reduction of Osterix+ cells in Yap1-KI iEC mutants (n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( G ) Decreased bone matrix protein Osteopontin (Opn, green) deposition in P21 Yap1-KI iEC femur relative to control. ECs, Emcn (red). ( H, I ) Representative μCT images of trabecular bone in P21 control and Yap1/Taz iΔEC femur ( H ). Quantitative analysis of trabecular volume (BV/TV, bone volume/total volume) trabecular (Tb.) number, Tb. thickness, and Tb. separation ( I ). (n = 5; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). Figure 7—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A ) Average body weight of P21 control and Yap1/Taz iΔEC mutants (n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( B, C ) Representative images of P21 control and Yap1/Taz iΔEC femur ( C ). Quantitation of femur length and weight show no changes between Yap1/Taz iΔEC mutants and littermate controls ( C ) (n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( D ) Confocal images showing Osterix+ cells (green) in relation to Emcn+ ECs (red) in P21 Yap1/Taz iΔEC and control metaphysis. Graph on the right shows quantitative analysis of Osterix+ (Osx+) cells (control n = 6 and Yap1/Taz iΔEC n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( E ) Confocal images showing bone vessels (Emcn), and the bone matrix protein Osteopontin in Yap1/Taz iΔEC vs control femur. ( F ) Representative confocal images of Osterix+ cells (green) in relation to Emcn+ ECs (red) in the P21 control and Yap1-KI iEC femoral metaphysis. Graph on the right shows significant reduction of Osterix+ cells in Yap1-KI iEC mutants (n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( G ) Decreased bone matrix protein Osteopontin (Opn, green) deposition in P21 Yap1-KI iEC femur relative to control. ECs, Emcn (red). ( H, I ) Representative μCT images of trabecular bone in P21 control and Yap1/Taz iΔEC femur ( H ). Quantitative analysis of trabecular volume (BV/TV, bone volume/total volume) trabecular (Tb.) number, Tb. thickness, and Tb. separation ( I ). (n = 5; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). Figure 7—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: Control, Two Tailed Test, Quantitation Assay

( A ) Quantifications of tibia weight and length show no changes between Yap1/Taz iΔEC mutants and littermate controls (n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( B ) Confocal images showing osteoblast markers collagen type aI (Col type I, alpha 1) and Osteocalcin (Ocn), osteoprogenitor differentiation marker Runx2 and the osteoclast marker ATP6V1B1 and two in Yap1/Taz iΔEC and control femur. ( C–E ) Average body weight of control and Yap1-KI iEC mice ( C ). Representative images of P21 control and Yap1-KI iEC femur ( D ). Quantification of femur and tibia weight and length in Yap1-KI iEC mutants relative to littermate controls ( E ) (n = 4, 5; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( F ) Representative confocal images of Osterix+ cells (red) in relation to Emcn+ ECs (blue) in P21 control, Yap1/Taz iΔEC and Yap1/Taz; Hif-1a iΔEC femoral metaphysis. Quantification of Osterix+ cells in Yap1/Taz iΔEC and Yap1/Taz; Hif-1a iΔEC mutants compared to control (n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). Figure 7—figure supplement 1—source data 1. Source data for .

Journal: eLife

Article Title: YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells

doi: 10.7554/eLife.50770

Figure Lengend Snippet: ( A ) Quantifications of tibia weight and length show no changes between Yap1/Taz iΔEC mutants and littermate controls (n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( B ) Confocal images showing osteoblast markers collagen type aI (Col type I, alpha 1) and Osteocalcin (Ocn), osteoprogenitor differentiation marker Runx2 and the osteoclast marker ATP6V1B1 and two in Yap1/Taz iΔEC and control femur. ( C–E ) Average body weight of control and Yap1-KI iEC mice ( C ). Representative images of P21 control and Yap1-KI iEC femur ( D ). Quantification of femur and tibia weight and length in Yap1-KI iEC mutants relative to littermate controls ( E ) (n = 4, 5; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). ( F ) Representative confocal images of Osterix+ cells (red) in relation to Emcn+ ECs (blue) in P21 control, Yap1/Taz iΔEC and Yap1/Taz; Hif-1a iΔEC femoral metaphysis. Quantification of Osterix+ cells in Yap1/Taz iΔEC and Yap1/Taz; Hif-1a iΔEC mutants compared to control (n = 6; data are presented as mean ±sem, P values, two-tailed unpaired t-test ). Figure 7—figure supplement 1—source data 1. Source data for .

Article Snippet: Quantitative PCR was carried out using gene TaqMan Gene Expression Master Mix (ThermoFisher Scientific, Cat#4369016) and specific Taqman probes human: eukaryotic 18S rRNA (4319413E), VEGFA (Hs00900055_m1, ANGPTL4 (Hs01101127_m1), IGFBP2 (Hs01040719_m1), XBP1 (Hs00231936_m1), CTGF (Hs01026927_g1), CYR61(Hs00998500_g1), YAP1(Hs00902712_g1), WWTR1(Hs00210007_m1), HIF1A(Hs00153153_m1) and mouse probes: Vegfa (Mm00437306_m1), Angptl4 (Mm00480431_m1), Ctgf (Mm01192932_g1), Cyr61 (Mm00487499_g1) from ThermoFisher Scientific (Cat# 4331182) using a C1000 Touch Thermal cycler (BIORAD).

Techniques: Two Tailed Test, Marker, Control

CPPs may impair endothelial mechanotransduction. ( A ) Western blotting measurements of mechanosensitive transcription factors KLF2, KLF4, NRF2, YAP1 and TAZ and phosphorylated forms of YAP1 and TAZ as compared to the expression of vimentin, CD31 and VE-cadherin in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to PBS group; ( B ) gene expression analysis of KLF2 , KLF4 , NFE2L2 , YAP1 , and WWTR1 genes in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. RT-qPCR measurements, the results are represented by a heat map. Gray and red colours mean fold change 0.51–1.99, and ≥2.00, respectively, compared to PBS group; ( C ) Western blotting measurements of mechanosensitive transcription factors Klf2, Klf4, Nrf2, Yap1, Taz and phosphorylated forms of Yap1 and Taz as compared to the expression of Cd31, Gapdh and histone H3 in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to NaCl group; ( D ) gene expression analysis of Klf2 , Klf4 , Nfe2l2 , Yap1 , and Wwtr1 genes in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). RT-qPCR measurements, the results are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.50, 0.51–1.99, and ≥2.00, respectively, compared to the NaCl group. HCAEC—human coronary artery endothelial cells, HITAEC—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPP—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, KLF—Krüppel-like factor, NRF—nuclear factor erythroid 2–related factor, YAP—Yes-associated protein, pYAP—phosphorylated YAP, TAZ—transcriptional co-activator with PDZ binding motif, pTAZ—phosphorylated TAZ, CD31—cluster of differentiation 31, VE-cadherin—vascular endothelial cadherin, NFE2L2—nuclear factor erythroid 2 like 2, WWTR—WW domain containing transcription regulator, Gapdh—glyceraldehyde 3-phosphate dehydrogenase, RT-qPCR—reverse transcription quantitative polymerase chain reaction.

Journal: International Journal of Molecular Sciences

Article Title: Calciprotein Particles Cause Endothelial Dysfunction under Flow

doi: 10.3390/ijms21228802

Figure Lengend Snippet: CPPs may impair endothelial mechanotransduction. ( A ) Western blotting measurements of mechanosensitive transcription factors KLF2, KLF4, NRF2, YAP1 and TAZ and phosphorylated forms of YAP1 and TAZ as compared to the expression of vimentin, CD31 and VE-cadherin in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to PBS group; ( B ) gene expression analysis of KLF2 , KLF4 , NFE2L2 , YAP1 , and WWTR1 genes in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. RT-qPCR measurements, the results are represented by a heat map. Gray and red colours mean fold change 0.51–1.99, and ≥2.00, respectively, compared to PBS group; ( C ) Western blotting measurements of mechanosensitive transcription factors Klf2, Klf4, Nrf2, Yap1, Taz and phosphorylated forms of Yap1 and Taz as compared to the expression of Cd31, Gapdh and histone H3 in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to NaCl group; ( D ) gene expression analysis of Klf2 , Klf4 , Nfe2l2 , Yap1 , and Wwtr1 genes in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). RT-qPCR measurements, the results are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.50, 0.51–1.99, and ≥2.00, respectively, compared to the NaCl group. HCAEC—human coronary artery endothelial cells, HITAEC—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPP—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, KLF—Krüppel-like factor, NRF—nuclear factor erythroid 2–related factor, YAP—Yes-associated protein, pYAP—phosphorylated YAP, TAZ—transcriptional co-activator with PDZ binding motif, pTAZ—phosphorylated TAZ, CD31—cluster of differentiation 31, VE-cadherin—vascular endothelial cadherin, NFE2L2—nuclear factor erythroid 2 like 2, WWTR—WW domain containing transcription regulator, Gapdh—glyceraldehyde 3-phosphate dehydrogenase, RT-qPCR—reverse transcription quantitative polymerase chain reaction.

Article Snippet: Blots were probed with rabbit antibodies to VCAM1 (ab134047, 1:1000, Abcam, Cambridge, UK), ICAM1 (ab109361, 1:1000, Abcam, Cambridge, UK), Snail and Slug (ab180714, 1:500, Abcam, Cambridge, UK), KLF4 (ab215036, 1:200, Abcam, Cambridge, UK), NRF2 (ab62352, 1:200, Abcam, Cambridge, UK), YAP1 (14074, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser109 (46931, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser127 (13008, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser397 (13619, 1:500, Cell Signaling Technology, Danvers, MA, USA), TAZ (70148, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-TAZ-Ser89 (59971, 1:200, Cell Signaling Technology, Danvers, MA, USA), vimentin (loading control, ab16700, 1:500, Abcam, Cambridge, UK) and VE-cadherin (loading control, 361900, 1:100, Thermo Fisher Scientific, Waltham, MA, USA), or mouse antibodies to N-cadherin (MA515633, 1:500, Thermo Fisher Scientific, Waltham, MA, USA), TWIST1 (sc-81417, 1:100, Santa Cruz Biotechnology, Dallas, TX, USA), KLF2 (NBP2-61812, 1:200, Novus Biologicals, Littleton, CO, USA), CD31 (loading control, ab9498, 1:1000, Abcam, Cambridge, UK), and GAPDH/histone H3 (loading control, ab139416, 1:250, Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Incubation, Gene Expression, Quantitative RT-PCR, Saline, Binding Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Sequences of customised primers for RT-qPCR.

Journal: International Journal of Molecular Sciences

Article Title: Calciprotein Particles Cause Endothelial Dysfunction under Flow

doi: 10.3390/ijms21228802

Figure Lengend Snippet: Sequences of customised primers for RT-qPCR.

Article Snippet: Blots were probed with rabbit antibodies to VCAM1 (ab134047, 1:1000, Abcam, Cambridge, UK), ICAM1 (ab109361, 1:1000, Abcam, Cambridge, UK), Snail and Slug (ab180714, 1:500, Abcam, Cambridge, UK), KLF4 (ab215036, 1:200, Abcam, Cambridge, UK), NRF2 (ab62352, 1:200, Abcam, Cambridge, UK), YAP1 (14074, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser109 (46931, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser127 (13008, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser397 (13619, 1:500, Cell Signaling Technology, Danvers, MA, USA), TAZ (70148, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-TAZ-Ser89 (59971, 1:200, Cell Signaling Technology, Danvers, MA, USA), vimentin (loading control, ab16700, 1:500, Abcam, Cambridge, UK) and VE-cadherin (loading control, 361900, 1:100, Thermo Fisher Scientific, Waltham, MA, USA), or mouse antibodies to N-cadherin (MA515633, 1:500, Thermo Fisher Scientific, Waltham, MA, USA), TWIST1 (sc-81417, 1:100, Santa Cruz Biotechnology, Dallas, TX, USA), KLF2 (NBP2-61812, 1:200, Novus Biologicals, Littleton, CO, USA), CD31 (loading control, ab9498, 1:1000, Abcam, Cambridge, UK), and GAPDH/histone H3 (loading control, ab139416, 1:250, Abcam, Cambridge, UK).

Techniques: Sequencing

CPPs may impair endothelial mechanotransduction. ( A ) Western blotting measurements of mechanosensitive transcription factors KLF2, KLF4, NRF2, YAP1 and TAZ and phosphorylated forms of YAP1 and TAZ as compared to the expression of vimentin, CD31 and VE-cadherin in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to PBS group; ( B ) gene expression analysis of KLF2 , KLF4 , NFE2L2 , YAP1 , and WWTR1 genes in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. RT-qPCR measurements, the results are represented by a heat map. Gray and red colours mean fold change 0.51–1.99, and ≥2.00, respectively, compared to PBS group; ( C ) Western blotting measurements of mechanosensitive transcription factors Klf2, Klf4, Nrf2, Yap1, Taz and phosphorylated forms of Yap1 and Taz as compared to the expression of Cd31, Gapdh and histone H3 in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to NaCl group; ( D ) gene expression analysis of Klf2 , Klf4 , Nfe2l2 , Yap1 , and Wwtr1 genes in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). RT-qPCR measurements, the results are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.50, 0.51–1.99, and ≥2.00, respectively, compared to the NaCl group. HCAEC—human coronary artery endothelial cells, HITAEC—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPP—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, KLF—Krüppel-like factor, NRF—nuclear factor erythroid 2–related factor, YAP—Yes-associated protein, pYAP—phosphorylated YAP, TAZ—transcriptional co-activator with PDZ binding motif, pTAZ—phosphorylated TAZ, CD31—cluster of differentiation 31, VE-cadherin—vascular endothelial cadherin, NFE2L2—nuclear factor erythroid 2 like 2, WWTR—WW domain containing transcription regulator, Gapdh—glyceraldehyde 3-phosphate dehydrogenase, RT-qPCR—reverse transcription quantitative polymerase chain reaction.

Journal: International Journal of Molecular Sciences

Article Title: Calciprotein Particles Cause Endothelial Dysfunction under Flow

doi: 10.3390/ijms21228802

Figure Lengend Snippet: CPPs may impair endothelial mechanotransduction. ( A ) Western blotting measurements of mechanosensitive transcription factors KLF2, KLF4, NRF2, YAP1 and TAZ and phosphorylated forms of YAP1 and TAZ as compared to the expression of vimentin, CD31 and VE-cadherin in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to PBS group; ( B ) gene expression analysis of KLF2 , KLF4 , NFE2L2 , YAP1 , and WWTR1 genes in HCAEC and HITAEC co-incubated with PBS, MPP, CPP-P or CPP-S in a flow system for 4 h. RT-qPCR measurements, the results are represented by a heat map. Gray and red colours mean fold change 0.51–1.99, and ≥2.00, respectively, compared to PBS group; ( C ) Western blotting measurements of mechanosensitive transcription factors Klf2, Klf4, Nrf2, Yap1, Taz and phosphorylated forms of Yap1 and Taz as compared to the expression of Cd31, Gapdh and histone H3 in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). Blot scans (left) and band densitometry analysis (right). The results of the latter are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.75, 0.76–1.24, and ≥1.25, respectively, compared to NaCl group; ( D ) gene expression analysis of Klf2 , Klf4 , Nfe2l2 , Yap1 , and Wwtr1 genes in the endothelial lysate collected from the descending aorta and aortic arch of Wistar rats which received consecutive tail vein injections of CPP-P, CPP-S or 0.9% NaCl (10 daily injections). RT-qPCR measurements, the results are represented by a heat map. Green, gray and red colours mean fold change ≤ 0.50, 0.51–1.99, and ≥2.00, respectively, compared to the NaCl group. HCAEC—human coronary artery endothelial cells, HITAEC—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPP—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, KLF—Krüppel-like factor, NRF—nuclear factor erythroid 2–related factor, YAP—Yes-associated protein, pYAP—phosphorylated YAP, TAZ—transcriptional co-activator with PDZ binding motif, pTAZ—phosphorylated TAZ, CD31—cluster of differentiation 31, VE-cadherin—vascular endothelial cadherin, NFE2L2—nuclear factor erythroid 2 like 2, WWTR—WW domain containing transcription regulator, Gapdh—glyceraldehyde 3-phosphate dehydrogenase, RT-qPCR—reverse transcription quantitative polymerase chain reaction.

Article Snippet: Blots were probed with rabbit antibodies to VCAM1 (ab134047, 1:1000, Abcam, Cambridge, UK), ICAM1 (ab109361, 1:1000, Abcam, Cambridge, UK), Snail and Slug (ab180714, 1:500, Abcam, Cambridge, UK), KLF4 (ab215036, 1:200, Abcam, Cambridge, UK), NRF2 (ab62352, 1:200, Abcam, Cambridge, UK), YAP1 (14074, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser109 (46931, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser127 (13008, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser397 (13619, 1:500, Cell Signaling Technology, Danvers, MA, USA), TAZ (70148, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-TAZ-Ser89 (59971, 1:200, Cell Signaling Technology, Danvers, MA, USA), vimentin (loading control, ab16700, 1:500, Abcam, Cambridge, UK) and VE-cadherin (loading control, 361900, 1:100, Thermo Fisher Scientific, Waltham, MA, USA), or mouse antibodies to N-cadherin (MA515633, 1:500, Thermo Fisher Scientific, Waltham, MA, USA), TWIST1 (sc-81417, 1:100, Santa Cruz Biotechnology, Dallas, TX, USA), KLF2 (NBP2-61812, 1:200, Novus Biologicals, Littleton, CO, USA), CD31 (loading control, ab9498, 1:1000, Abcam, Cambridge, UK), and GAPDH/histone H3 (loading control, ab139416, 1:250, Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Incubation, Gene Expression, Quantitative RT-PCR, Saline, Binding Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Sequences of customised primers for RT-qPCR.

Journal: International Journal of Molecular Sciences

Article Title: Calciprotein Particles Cause Endothelial Dysfunction under Flow

doi: 10.3390/ijms21228802

Figure Lengend Snippet: Sequences of customised primers for RT-qPCR.

Article Snippet: Blots were probed with rabbit antibodies to VCAM1 (ab134047, 1:1000, Abcam, Cambridge, UK), ICAM1 (ab109361, 1:1000, Abcam, Cambridge, UK), Snail and Slug (ab180714, 1:500, Abcam, Cambridge, UK), KLF4 (ab215036, 1:200, Abcam, Cambridge, UK), NRF2 (ab62352, 1:200, Abcam, Cambridge, UK), YAP1 (14074, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser109 (46931, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser127 (13008, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-YAP1-Ser397 (13619, 1:500, Cell Signaling Technology, Danvers, MA, USA), TAZ (70148, 1:500, Cell Signaling Technology, Danvers, MA, USA), phospho-TAZ-Ser89 (59971, 1:200, Cell Signaling Technology, Danvers, MA, USA), vimentin (loading control, ab16700, 1:500, Abcam, Cambridge, UK) and VE-cadherin (loading control, 361900, 1:100, Thermo Fisher Scientific, Waltham, MA, USA), or mouse antibodies to N-cadherin (MA515633, 1:500, Thermo Fisher Scientific, Waltham, MA, USA), TWIST1 (sc-81417, 1:100, Santa Cruz Biotechnology, Dallas, TX, USA), KLF2 (NBP2-61812, 1:200, Novus Biologicals, Littleton, CO, USA), CD31 (loading control, ab9498, 1:1000, Abcam, Cambridge, UK), and GAPDH/histone H3 (loading control, ab139416, 1:250, Abcam, Cambridge, UK).

Techniques: Sequencing

Development of cataracts in the Yap1 +/ − mice. ( A , B ) Images of WT eye ( A ) and the cataract eye of Yap1 +/ − mouse ( B ) at two months of age. ( C , D ) Transparency of freshly collected lenses. The lenses were placed on the grid papers and photographed by transillumination with white light. Transparency is indicated by the clarity of grid. ( E ) Percentage of the animals at each age group that developed cataracts. Note that each age group is a separate cohort of mice. ( F , G ) Histological hematoxylin and eosin staining of paraffin-embedded eye sections from WT and Yap1 +/ − cataract mice at three months old. The cortical rupture in Yap1 +/ − lens was noticeable in G . ( F1 , G1 ) High-magnification views of F and G, respectively. Scale bar : 500 µm ( A – D , F , and G ), 50 µm ( F1 and G1 ).

Journal: Investigative Ophthalmology & Visual Science

Article Title: Heterozygous Loss of Yap1 in Mice Causes Progressive Cataracts

doi: 10.1167/iovs.61.12.21

Figure Lengend Snippet: Development of cataracts in the Yap1 +/ − mice. ( A , B ) Images of WT eye ( A ) and the cataract eye of Yap1 +/ − mouse ( B ) at two months of age. ( C , D ) Transparency of freshly collected lenses. The lenses were placed on the grid papers and photographed by transillumination with white light. Transparency is indicated by the clarity of grid. ( E ) Percentage of the animals at each age group that developed cataracts. Note that each age group is a separate cohort of mice. ( F , G ) Histological hematoxylin and eosin staining of paraffin-embedded eye sections from WT and Yap1 +/ − cataract mice at three months old. The cortical rupture in Yap1 +/ − lens was noticeable in G . ( F1 , G1 ) High-magnification views of F and G, respectively. Scale bar : 500 µm ( A – D , F , and G ), 50 µm ( F1 and G1 ).

Article Snippet: The primary antibodies used in this study were mouse anti-YAP1 (1:100, cat. no. 56701; Abcam, Cambridge, MA, USA), mouse anti YAP1 (1:100, cat. no. sc-101199; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-β-catenin (1:100, cat. no. BD 610154; BD Bioscience, Franklin Lakes, NJ, USA), rabbit anti-β-catenin (D10A8) (1; 100, cat. no. 8480S; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ZO-1 (1:200, cat. no. 61-7300; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-βB1-crystallin (1:200, cat. no. NBP2-68576; Novus Biologicals, Centennial, CO, USA), rabbit anti-Crim1 (1:100, cat. no. AB5699, Millipore Sigma, Burlington, MA, USA), rat anti-BrdU (1:800, MAS 250c, Harlan-Sera Lab, Belton Loughborough, Leicestershire, UK).

Techniques: Staining

Reduction of lens size, LEC density, and LEC proliferation in Yap1 +/ − mice. Experiments were performed on one-month-old WT and Yap1 +/ − sibling mice. ( A , B ) Images of the freshly isolated lenses from WT and Yap1 +/ − mice show difference in lens size. ( C ) Quantification of relative lens size based on the diameter of lens (n = 8 mice for each group). ( D , E ) Hematoxylin and eosin staining of eye sections from WT and Yap1 +/ − mice. ( D1 – E1 ) High-magnification views of D and E, respectively. ( F , G ) Fluorescent images taken from the central zone (CZ) of flat-mounted lens epithelium stained with DAPI to identify cell nuclei. ( H ) Relative cell density calculated by the number of cell nuclei per equal unit of area using Image J software (n = 5 mice for each group). ( I , J ) Fluorescent images taken from the germinative zone (GZ) and transitional zone (TZ) of lens immunostained with anti-BrdU antibody ( red ) and nuclear counterstained with DAPI ( blue ). ( K ) Relative number of BrdU + cells in WT and Yap1 +/ − mice. The quantitative analysis of representative images was conducted using Image J software. The numbers of BrdU + cell were normalized by the distance along equatorial plane (n = 6 mice for each group). * P < 0.05. *** P < 0.005. Scale bar: 500 µm ( A , B , D , and E ), 50 µm ( D1 , E1 , F , G , I , and J ). * indicates large breakdown of lens epithelium. All values are expressed as the mean ± SD.

Journal: Investigative Ophthalmology & Visual Science

Article Title: Heterozygous Loss of Yap1 in Mice Causes Progressive Cataracts

doi: 10.1167/iovs.61.12.21

Figure Lengend Snippet: Reduction of lens size, LEC density, and LEC proliferation in Yap1 +/ − mice. Experiments were performed on one-month-old WT and Yap1 +/ − sibling mice. ( A , B ) Images of the freshly isolated lenses from WT and Yap1 +/ − mice show difference in lens size. ( C ) Quantification of relative lens size based on the diameter of lens (n = 8 mice for each group). ( D , E ) Hematoxylin and eosin staining of eye sections from WT and Yap1 +/ − mice. ( D1 – E1 ) High-magnification views of D and E, respectively. ( F , G ) Fluorescent images taken from the central zone (CZ) of flat-mounted lens epithelium stained with DAPI to identify cell nuclei. ( H ) Relative cell density calculated by the number of cell nuclei per equal unit of area using Image J software (n = 5 mice for each group). ( I , J ) Fluorescent images taken from the germinative zone (GZ) and transitional zone (TZ) of lens immunostained with anti-BrdU antibody ( red ) and nuclear counterstained with DAPI ( blue ). ( K ) Relative number of BrdU + cells in WT and Yap1 +/ − mice. The quantitative analysis of representative images was conducted using Image J software. The numbers of BrdU + cell were normalized by the distance along equatorial plane (n = 6 mice for each group). * P < 0.05. *** P < 0.005. Scale bar: 500 µm ( A , B , D , and E ), 50 µm ( D1 , E1 , F , G , I , and J ). * indicates large breakdown of lens epithelium. All values are expressed as the mean ± SD.

Article Snippet: The primary antibodies used in this study were mouse anti-YAP1 (1:100, cat. no. 56701; Abcam, Cambridge, MA, USA), mouse anti YAP1 (1:100, cat. no. sc-101199; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-β-catenin (1:100, cat. no. BD 610154; BD Bioscience, Franklin Lakes, NJ, USA), rabbit anti-β-catenin (D10A8) (1; 100, cat. no. 8480S; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ZO-1 (1:200, cat. no. 61-7300; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-βB1-crystallin (1:200, cat. no. NBP2-68576; Novus Biologicals, Centennial, CO, USA), rabbit anti-Crim1 (1:100, cat. no. AB5699, Millipore Sigma, Burlington, MA, USA), rat anti-BrdU (1:800, MAS 250c, Harlan-Sera Lab, Belton Loughborough, Leicestershire, UK).

Techniques: Isolation, Staining, Software

No apoptosis induction in Yap1 +/ − lens epithelium. Experiments were performed on one-month-old WT and Yap1 +/ − sibling mice. Six mice from each group and six sections from each eye were examined. ( A, B ) Representative images of TUNEL assay staining on the lens sections of WT ( A ) and Yap1 +/ − ( B ) mice. Scale bar: 50 µm, arrow heads indicate the apoptotic cells detected by TUNEL in iris tissue.

Journal: Investigative Ophthalmology & Visual Science

Article Title: Heterozygous Loss of Yap1 in Mice Causes Progressive Cataracts

doi: 10.1167/iovs.61.12.21

Figure Lengend Snippet: No apoptosis induction in Yap1 +/ − lens epithelium. Experiments were performed on one-month-old WT and Yap1 +/ − sibling mice. Six mice from each group and six sections from each eye were examined. ( A, B ) Representative images of TUNEL assay staining on the lens sections of WT ( A ) and Yap1 +/ − ( B ) mice. Scale bar: 50 µm, arrow heads indicate the apoptotic cells detected by TUNEL in iris tissue.

Article Snippet: The primary antibodies used in this study were mouse anti-YAP1 (1:100, cat. no. 56701; Abcam, Cambridge, MA, USA), mouse anti YAP1 (1:100, cat. no. sc-101199; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-β-catenin (1:100, cat. no. BD 610154; BD Bioscience, Franklin Lakes, NJ, USA), rabbit anti-β-catenin (D10A8) (1; 100, cat. no. 8480S; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ZO-1 (1:200, cat. no. 61-7300; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-βB1-crystallin (1:200, cat. no. NBP2-68576; Novus Biologicals, Centennial, CO, USA), rabbit anti-Crim1 (1:100, cat. no. AB5699, Millipore Sigma, Burlington, MA, USA), rat anti-BrdU (1:800, MAS 250c, Harlan-Sera Lab, Belton Loughborough, Leicestershire, UK).

Techniques: TUNEL Assay, Staining

Disruption of cell-cell junctions in Yap1 +/ − lens epithelium. ( A , B ) Representative images of flat-mounted lens epithelium stained with phalloidin and DAPI. ( C1 – C4 , D1 – D4 , E1 – E4 ) The representative images at anterior of WT ( C1 – C4 ) and Yap1 +/ − ( D1 – D4 , E1 – E4 ) lenses immunostained with antibodies against β-catenin and ZO-1, with DAPI counterstaining to show cell nuclei. ( F1–F4 , G1–G4 ) The representative images at equator of WT ( F1 - F4 ) and Yap1 +/ − ( G1 – G4 ) lenses immunostained with antibodies against β-catenin and ZO-1, and DAPI to counterstained nuclei. Six mice per group were used for each experiment.

Journal: Investigative Ophthalmology & Visual Science

Article Title: Heterozygous Loss of Yap1 in Mice Causes Progressive Cataracts

doi: 10.1167/iovs.61.12.21

Figure Lengend Snippet: Disruption of cell-cell junctions in Yap1 +/ − lens epithelium. ( A , B ) Representative images of flat-mounted lens epithelium stained with phalloidin and DAPI. ( C1 – C4 , D1 – D4 , E1 – E4 ) The representative images at anterior of WT ( C1 – C4 ) and Yap1 +/ − ( D1 – D4 , E1 – E4 ) lenses immunostained with antibodies against β-catenin and ZO-1, with DAPI counterstaining to show cell nuclei. ( F1–F4 , G1–G4 ) The representative images at equator of WT ( F1 - F4 ) and Yap1 +/ − ( G1 – G4 ) lenses immunostained with antibodies against β-catenin and ZO-1, and DAPI to counterstained nuclei. Six mice per group were used for each experiment.

Article Snippet: The primary antibodies used in this study were mouse anti-YAP1 (1:100, cat. no. 56701; Abcam, Cambridge, MA, USA), mouse anti YAP1 (1:100, cat. no. sc-101199; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-β-catenin (1:100, cat. no. BD 610154; BD Bioscience, Franklin Lakes, NJ, USA), rabbit anti-β-catenin (D10A8) (1; 100, cat. no. 8480S; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ZO-1 (1:200, cat. no. 61-7300; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-βB1-crystallin (1:200, cat. no. NBP2-68576; Novus Biologicals, Centennial, CO, USA), rabbit anti-Crim1 (1:100, cat. no. AB5699, Millipore Sigma, Burlington, MA, USA), rat anti-BrdU (1:800, MAS 250c, Harlan-Sera Lab, Belton Loughborough, Leicestershire, UK).

Techniques: Disruption, Staining

Disrupted lens fiber morphology and normal βB1-crystallin expression in Yap1 +/ − lens. Experiments were performed on one-month-old WT and Yap1 +/ − sibling mice. Six mice from each group and at least six transverse sections around the germinative zone from each eye were examined. ( A , B ) Representative images of transverse sections of lenses immunostained with antibodies against β-catenin and ZO-1 with nuclear counter staining by DAPI. ( C , D ) Representative images of transverse sections of lenses immunostained with antibodies against βB1-crystallin, β-catenin, with DAPI nuclear counter-staining to show cell nuclei. All images are in the same magnification. Scale bar: 50 µm.

Journal: Investigative Ophthalmology & Visual Science

Article Title: Heterozygous Loss of Yap1 in Mice Causes Progressive Cataracts

doi: 10.1167/iovs.61.12.21

Figure Lengend Snippet: Disrupted lens fiber morphology and normal βB1-crystallin expression in Yap1 +/ − lens. Experiments were performed on one-month-old WT and Yap1 +/ − sibling mice. Six mice from each group and at least six transverse sections around the germinative zone from each eye were examined. ( A , B ) Representative images of transverse sections of lenses immunostained with antibodies against β-catenin and ZO-1 with nuclear counter staining by DAPI. ( C , D ) Representative images of transverse sections of lenses immunostained with antibodies against βB1-crystallin, β-catenin, with DAPI nuclear counter-staining to show cell nuclei. All images are in the same magnification. Scale bar: 50 µm.

Article Snippet: The primary antibodies used in this study were mouse anti-YAP1 (1:100, cat. no. 56701; Abcam, Cambridge, MA, USA), mouse anti YAP1 (1:100, cat. no. sc-101199; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-β-catenin (1:100, cat. no. BD 610154; BD Bioscience, Franklin Lakes, NJ, USA), rabbit anti-β-catenin (D10A8) (1; 100, cat. no. 8480S; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ZO-1 (1:200, cat. no. 61-7300; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-βB1-crystallin (1:200, cat. no. NBP2-68576; Novus Biologicals, Centennial, CO, USA), rabbit anti-Crim1 (1:100, cat. no. AB5699, Millipore Sigma, Burlington, MA, USA), rat anti-BrdU (1:800, MAS 250c, Harlan-Sera Lab, Belton Loughborough, Leicestershire, UK).

Techniques: Expressing, Staining

YAP1 expression in WT and Yap1 +/ − lens epithelia. ( A – A1 , B – B1 ) Immunostaining of YAP1 ( red ) and counter-stained with DAPI ( blue ) on anterior ( A–A1 ) and equator ( B–B1 ) of the WT lenses. Scale bar: 50 µm. ( C ) qPCR analysis of Yap1 mRNA in the lens epithelium of WT and Yap1 +/ − eyes, showing a significant reduced Yap1 mRNA in the heterozygous mutant lenses. The comparative threshold cycle (CT) methods normalized to Actb were used to analyze relative changes in gene expression. The relative expression (arbitrary units) is expressed as a ratio (n = 3 independent samples). Each total RNA sample was isolated from lens epithelial cells pooled from five to eight mice (10 – 16 lenses), and qPCR analysis for each sample was performed in triplicate. * P < 0.05. ( D ) Representative Western blotting shows YAP1 protein expression level in the WT and Yap1 +/ − lens epithelia. Alpha-TUBULIN is used as loading control. ( E ) The quantification of relative YAP1 protein level was normalized to α-TUBULIN protein level after quantification of Western blot images using Image J software (n = 3). Each sample was pooled from five to eight mice, * P < 0.05. All values are expressed as the mean ± SD.

Journal: Investigative Ophthalmology & Visual Science

Article Title: Heterozygous Loss of Yap1 in Mice Causes Progressive Cataracts

doi: 10.1167/iovs.61.12.21

Figure Lengend Snippet: YAP1 expression in WT and Yap1 +/ − lens epithelia. ( A – A1 , B – B1 ) Immunostaining of YAP1 ( red ) and counter-stained with DAPI ( blue ) on anterior ( A–A1 ) and equator ( B–B1 ) of the WT lenses. Scale bar: 50 µm. ( C ) qPCR analysis of Yap1 mRNA in the lens epithelium of WT and Yap1 +/ − eyes, showing a significant reduced Yap1 mRNA in the heterozygous mutant lenses. The comparative threshold cycle (CT) methods normalized to Actb were used to analyze relative changes in gene expression. The relative expression (arbitrary units) is expressed as a ratio (n = 3 independent samples). Each total RNA sample was isolated from lens epithelial cells pooled from five to eight mice (10 – 16 lenses), and qPCR analysis for each sample was performed in triplicate. * P < 0.05. ( D ) Representative Western blotting shows YAP1 protein expression level in the WT and Yap1 +/ − lens epithelia. Alpha-TUBULIN is used as loading control. ( E ) The quantification of relative YAP1 protein level was normalized to α-TUBULIN protein level after quantification of Western blot images using Image J software (n = 3). Each sample was pooled from five to eight mice, * P < 0.05. All values are expressed as the mean ± SD.

Article Snippet: The primary antibodies used in this study were mouse anti-YAP1 (1:100, cat. no. 56701; Abcam, Cambridge, MA, USA), mouse anti YAP1 (1:100, cat. no. sc-101199; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-β-catenin (1:100, cat. no. BD 610154; BD Bioscience, Franklin Lakes, NJ, USA), rabbit anti-β-catenin (D10A8) (1; 100, cat. no. 8480S; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ZO-1 (1:200, cat. no. 61-7300; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-βB1-crystallin (1:200, cat. no. NBP2-68576; Novus Biologicals, Centennial, CO, USA), rabbit anti-Crim1 (1:100, cat. no. AB5699, Millipore Sigma, Burlington, MA, USA), rat anti-BrdU (1:800, MAS 250c, Harlan-Sera Lab, Belton Loughborough, Leicestershire, UK).

Techniques: Expressing, Immunostaining, Staining, Mutagenesis, Gene Expression, Isolation, Western Blot, Control, Software

Reduced Crim1 expression in Yap1 +/ − lens epithelium. ( A ) The qPCR quantification of Crim1 mRNA from the WT and Yap1 +/ − lens epithelia. The comparative threshold cycle methods normalized to Actb were used to analyze relative changes in gene expression. The relative expression (arbitrary units) is presented as a ratio. Data are expressed as mean ± the SD (n = 3 independent samples), each sample was total RNA isolated from lens epithelium pooled from five to eight mice (10 – 16 lenses), and qPCR analysis for each sample was performed in triplicate. * P < 0.05. ( B ) Immunostaining with anti-CRIM1 antibody ( red ) and DAPI ( blue ) on WT and Yap1 +/ − lens epitheliums. Scale bar: 50 µm. ( C ) Representative Western blotting on the protein samples from WT and Yap1 +/ − lens epithelia. Alpha-TUBULIN is used as loading control. ( D ) The quantification of relative CRIM protein level against α-TUBULIN protein from Western blot images using Image J software (n = 3). Each sample was pooled from five to eight mice. *** P < 0.005. ( E ) Western blot performed on the protein samples from the WT and Yap1 +/ − lens epithelia using indicated antibodies. ( F ) Relative FAK phosphorylation level normalized to the FAK protein level, and ERK1/2 phosphorylation signal relative to ERK1/2 total protein signal, were quantitated from Western blot images using Image J software (n = 3). Each sample was pooled from five to eight mice. * P < 0.05, *** P < 0.005. All values are expressed as the mean ± SD.

Journal: Investigative Ophthalmology & Visual Science

Article Title: Heterozygous Loss of Yap1 in Mice Causes Progressive Cataracts

doi: 10.1167/iovs.61.12.21

Figure Lengend Snippet: Reduced Crim1 expression in Yap1 +/ − lens epithelium. ( A ) The qPCR quantification of Crim1 mRNA from the WT and Yap1 +/ − lens epithelia. The comparative threshold cycle methods normalized to Actb were used to analyze relative changes in gene expression. The relative expression (arbitrary units) is presented as a ratio. Data are expressed as mean ± the SD (n = 3 independent samples), each sample was total RNA isolated from lens epithelium pooled from five to eight mice (10 – 16 lenses), and qPCR analysis for each sample was performed in triplicate. * P < 0.05. ( B ) Immunostaining with anti-CRIM1 antibody ( red ) and DAPI ( blue ) on WT and Yap1 +/ − lens epitheliums. Scale bar: 50 µm. ( C ) Representative Western blotting on the protein samples from WT and Yap1 +/ − lens epithelia. Alpha-TUBULIN is used as loading control. ( D ) The quantification of relative CRIM protein level against α-TUBULIN protein from Western blot images using Image J software (n = 3). Each sample was pooled from five to eight mice. *** P < 0.005. ( E ) Western blot performed on the protein samples from the WT and Yap1 +/ − lens epithelia using indicated antibodies. ( F ) Relative FAK phosphorylation level normalized to the FAK protein level, and ERK1/2 phosphorylation signal relative to ERK1/2 total protein signal, were quantitated from Western blot images using Image J software (n = 3). Each sample was pooled from five to eight mice. * P < 0.05, *** P < 0.005. All values are expressed as the mean ± SD.

Article Snippet: The primary antibodies used in this study were mouse anti-YAP1 (1:100, cat. no. 56701; Abcam, Cambridge, MA, USA), mouse anti YAP1 (1:100, cat. no. sc-101199; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-β-catenin (1:100, cat. no. BD 610154; BD Bioscience, Franklin Lakes, NJ, USA), rabbit anti-β-catenin (D10A8) (1; 100, cat. no. 8480S; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ZO-1 (1:200, cat. no. 61-7300; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-βB1-crystallin (1:200, cat. no. NBP2-68576; Novus Biologicals, Centennial, CO, USA), rabbit anti-Crim1 (1:100, cat. no. AB5699, Millipore Sigma, Burlington, MA, USA), rat anti-BrdU (1:800, MAS 250c, Harlan-Sera Lab, Belton Loughborough, Leicestershire, UK).

Techniques: Expressing, Gene Expression, Isolation, Immunostaining, Western Blot, Control, Software, Phospho-proteomics

Rescue of the proliferation phenotype by overexpression of Crim1 in the primary Yap1 +/ − LECs. ( A–D ) The representative images of WT ( A , B ) and Yap1 +/ − ( C , D ) LECs transduced by lentiviral control vector ( A , C ) or lentiviral Crim1-expressing vector ( B , D ). Two days after transduction, the LEC culture medium was replaced with warm and fresh prepared medium containing 10 µM BrdU for two hours. After brief rinsing with phosphate-buffered saline solution, the LEC was fixed and processed for anti-BrdU immunostaining ( red ), and nuclei were counterstained with DAPI ( blue ). Scale bar: 100 µm. ( E ) Quantification of BrdU-positive cells in the indicated groups. Data are expressed as mean ± SD, n = 3 repeats. * P ≤ 0.05, ** P ≤ 0.01. ( F ) Western blot analysis of the YAP1, CRIM1, and α-TUBULIN protein levels in the indicated samples as ( A–D ).

Journal: Investigative Ophthalmology & Visual Science

Article Title: Heterozygous Loss of Yap1 in Mice Causes Progressive Cataracts

doi: 10.1167/iovs.61.12.21

Figure Lengend Snippet: Rescue of the proliferation phenotype by overexpression of Crim1 in the primary Yap1 +/ − LECs. ( A–D ) The representative images of WT ( A , B ) and Yap1 +/ − ( C , D ) LECs transduced by lentiviral control vector ( A , C ) or lentiviral Crim1-expressing vector ( B , D ). Two days after transduction, the LEC culture medium was replaced with warm and fresh prepared medium containing 10 µM BrdU for two hours. After brief rinsing with phosphate-buffered saline solution, the LEC was fixed and processed for anti-BrdU immunostaining ( red ), and nuclei were counterstained with DAPI ( blue ). Scale bar: 100 µm. ( E ) Quantification of BrdU-positive cells in the indicated groups. Data are expressed as mean ± SD, n = 3 repeats. * P ≤ 0.05, ** P ≤ 0.01. ( F ) Western blot analysis of the YAP1, CRIM1, and α-TUBULIN protein levels in the indicated samples as ( A–D ).

Article Snippet: The primary antibodies used in this study were mouse anti-YAP1 (1:100, cat. no. 56701; Abcam, Cambridge, MA, USA), mouse anti YAP1 (1:100, cat. no. sc-101199; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-β-catenin (1:100, cat. no. BD 610154; BD Bioscience, Franklin Lakes, NJ, USA), rabbit anti-β-catenin (D10A8) (1; 100, cat. no. 8480S; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-ZO-1 (1:200, cat. no. 61-7300; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-βB1-crystallin (1:200, cat. no. NBP2-68576; Novus Biologicals, Centennial, CO, USA), rabbit anti-Crim1 (1:100, cat. no. AB5699, Millipore Sigma, Burlington, MA, USA), rat anti-BrdU (1:800, MAS 250c, Harlan-Sera Lab, Belton Loughborough, Leicestershire, UK).

Techniques: Over Expression, Control, Plasmid Preparation, Expressing, Transduction, Saline, Immunostaining, Western Blot

a, Schematic diagram shows different stages of T cell development. Hematopoietic stem and progenitor cells (HSPC), double negative (DN), double positive (DP), single positive (SP), and regulatory T cells (Treg). Representative flow cytometry plots (right) show cKit, CD25, and Bcl11b expression patterns in distinct DN populations. Note: “DN1” throughout refers only to cKit high DN1, also known as Early T Progenitor (ETP) cells. Flow cytometry data was obtained from artificial thymic organoid (ATO) culture on day 9. b, Runx1 and Runx3 occupancy patterns in non-promoter regions of indicated cell populations are shown as peak-centered heatmaps. Runx binding profiles in DN1 and DN3 were detected by C&R from 2 independent experiments (merged data shown), and others were reported using ChIP-seq , – . MK, Maturing megakaryocytes. Numbers indicate the percent of group among total Runx binding sites. c, Diagram illustrates the key marker gene expression patterns in early T-cell development with associated levels of Runx1+Runx3 protein (left). Different groups of non-promoter Runx binding sites in early T-development are defined (right). d, Runx1 and Runx3 occupancy patterns in Phase 1 and Phase 2 cells are shown together with ATAC and H3K4me2 profiles , . Stage-specific Runx binding groups were determined by C&R using DN1 (cKit hi CD25 - ) cells obtained from in vitro OP9-Dll1 culture and thymic DN3 (cKit low CD44 low CD25 + ) cells. e, Representative UCSC genome browser tracks for Runx C&R (independent replicates), and published ATAC-seq, and ChIP-seq data for H3K4me2, H3K27me3, CTCF, and SMC3 are shown , , . Chromatin states computed by ChromHMM are displayed as a colormap at the bottom. P1, P2: Phase 1, Phase 2. Representative Phase 1-preferential Runx binding sites (near Meis1 , left) and Phase 2-preferential sites (near Ets1 , right) are displayed. f, g, Motif analysis was conducted within constantly open chromatin sites possessing different developmental patterns of Runx binding. f, Runx motif frequency within a peak (left) and the best Runx motif quality (position weight matrix score) within each peak (right) are shown. The dotted horizontal line on the violin plot indicates threshold motif quality to score as possessing Runx motif. Thin vertical black lines mark 1.5x interquartile range and thick vertical black lines show interquartile range. Red bars with white circles indicate median values. Two sample Kolmogorov-Smirnov (KS) tests, comparing each to Group3 motif scores: *** p<0.001. g, Motif frequencies of PU.1, E2A, and ETS factors in each Runx binding site Group are displayed.

Journal: bioRxiv

Article Title: Runx factors launch T-cell and innate lymphoid programs via direct and gene network-based mechanisms

doi: 10.1101/2022.11.18.517146

Figure Lengend Snippet: a, Schematic diagram shows different stages of T cell development. Hematopoietic stem and progenitor cells (HSPC), double negative (DN), double positive (DP), single positive (SP), and regulatory T cells (Treg). Representative flow cytometry plots (right) show cKit, CD25, and Bcl11b expression patterns in distinct DN populations. Note: “DN1” throughout refers only to cKit high DN1, also known as Early T Progenitor (ETP) cells. Flow cytometry data was obtained from artificial thymic organoid (ATO) culture on day 9. b, Runx1 and Runx3 occupancy patterns in non-promoter regions of indicated cell populations are shown as peak-centered heatmaps. Runx binding profiles in DN1 and DN3 were detected by C&R from 2 independent experiments (merged data shown), and others were reported using ChIP-seq , – . MK, Maturing megakaryocytes. Numbers indicate the percent of group among total Runx binding sites. c, Diagram illustrates the key marker gene expression patterns in early T-cell development with associated levels of Runx1+Runx3 protein (left). Different groups of non-promoter Runx binding sites in early T-development are defined (right). d, Runx1 and Runx3 occupancy patterns in Phase 1 and Phase 2 cells are shown together with ATAC and H3K4me2 profiles , . Stage-specific Runx binding groups were determined by C&R using DN1 (cKit hi CD25 - ) cells obtained from in vitro OP9-Dll1 culture and thymic DN3 (cKit low CD44 low CD25 + ) cells. e, Representative UCSC genome browser tracks for Runx C&R (independent replicates), and published ATAC-seq, and ChIP-seq data for H3K4me2, H3K27me3, CTCF, and SMC3 are shown , , . Chromatin states computed by ChromHMM are displayed as a colormap at the bottom. P1, P2: Phase 1, Phase 2. Representative Phase 1-preferential Runx binding sites (near Meis1 , left) and Phase 2-preferential sites (near Ets1 , right) are displayed. f, g, Motif analysis was conducted within constantly open chromatin sites possessing different developmental patterns of Runx binding. f, Runx motif frequency within a peak (left) and the best Runx motif quality (position weight matrix score) within each peak (right) are shown. The dotted horizontal line on the violin plot indicates threshold motif quality to score as possessing Runx motif. Thin vertical black lines mark 1.5x interquartile range and thick vertical black lines show interquartile range. Red bars with white circles indicate median values. Two sample Kolmogorov-Smirnov (KS) tests, comparing each to Group3 motif scores: *** p<0.001. g, Motif frequencies of PU.1, E2A, and ETS factors in each Runx binding site Group are displayed.

Article Snippet: Three gRNAs were utilized to target each Runx paralog (Addgene #189799, #189800, #189801, #189802, #189803, #189804, #189805, #189806).

Techniques: Flow Cytometry, Expressing, Binding Assay, ChIP-sequencing, Marker, Gene Expression, In Vitro

a, Experimental design for testing Runx dosage effect on early T-development is displayed. b, Representative histograms show intracellular Runx1 protein levels detected by flow cytometry in control or Runx1-overexpression (OE) vector-transduced Phase 1 cells. Numbers indicate geometric mean fluorescence intensities (gMFI) of Runx1. Graph summarizes results from 8 independent experiments. Comparisons by two-way ANOVA. c-d, Flow cytometry data show cKit, CD44, CD25, Bcl11b-mCitrine reporter levels after delivering empty control or Runx1 overexpression (OE) vectors on day 2 ( c, top) or day 3 ( c, bottom) of T-cell development. Graphs in d , summarize mean values from 6-10 independent experiments with standard deviation (error bar). Comparisons by t-test. e, Histograms display protein expression levels of TCF1, GATA3, and PU.1 at day 2 and day 4 after Runx1 overexpression in Phase 1. Phase 1 (live, alternative lineage - , cKit high ) cells were separated as CD25 - DN1 and CD25 + DN2 populations to compare target protein levels. Graphs display mean values from 5-7 independent experiments with standard deviations. Comparisons by two-way ANOVA. ***=p-value<0.001, **=p- value<0.01, *=p-value<0.05, ns=not significant.

Journal: bioRxiv

Article Title: Runx factors launch T-cell and innate lymphoid programs via direct and gene network-based mechanisms

doi: 10.1101/2022.11.18.517146

Figure Lengend Snippet: a, Experimental design for testing Runx dosage effect on early T-development is displayed. b, Representative histograms show intracellular Runx1 protein levels detected by flow cytometry in control or Runx1-overexpression (OE) vector-transduced Phase 1 cells. Numbers indicate geometric mean fluorescence intensities (gMFI) of Runx1. Graph summarizes results from 8 independent experiments. Comparisons by two-way ANOVA. c-d, Flow cytometry data show cKit, CD44, CD25, Bcl11b-mCitrine reporter levels after delivering empty control or Runx1 overexpression (OE) vectors on day 2 ( c, top) or day 3 ( c, bottom) of T-cell development. Graphs in d , summarize mean values from 6-10 independent experiments with standard deviation (error bar). Comparisons by t-test. e, Histograms display protein expression levels of TCF1, GATA3, and PU.1 at day 2 and day 4 after Runx1 overexpression in Phase 1. Phase 1 (live, alternative lineage - , cKit high ) cells were separated as CD25 - DN1 and CD25 + DN2 populations to compare target protein levels. Graphs display mean values from 5-7 independent experiments with standard deviations. Comparisons by two-way ANOVA. ***=p-value<0.001, **=p- value<0.01, *=p-value<0.05, ns=not significant.

Article Snippet: Three gRNAs were utilized to target each Runx paralog (Addgene #189799, #189800, #189801, #189802, #189803, #189804, #189805, #189806).

Techniques: Flow Cytometry, Control, Over Expression, Plasmid Preparation, Fluorescence, Standard Deviation, Expressing

a, Experimental schematics for single cell RNA-seq (scRNA-seq) are depicted. Each experimental condition group was marked by a different hashtag oligo (HTO) and equal numbers of cells from each were pooled for scRNA-seq. Two independent scRNA-seq experiments were performed. See Methods for details. b-d, UMAP2-3 illustrate scRNA- seq data from Runx1 OE and Runx1/Runx3 double knockout (KO) in Phase 1. b, Color intensity in UMAP displays expression levels of indicated genes, which are informative to represent different T-development stages. c, Types of Runx perturbation (control, Runx1 OE, and Runx1/Runx3 KO cells) with cells from different experimental time points are highlighted in UMAP2-3 space. d, Cells from scRNA-seq are colored by Louvain clusters. e, Area-proportional Venn diagram shows the number of Runx-activated (blue) and - inhibited genes (orange) in OE and/or KO perturbations. The common, core target gene numbers are shown in red. f, Scatter plots compare Log 2 fold-changes of Runx target gene expression in Runx1 OE and Runx KO conditions at d2-d3 (left) or d4-d6 (right) after Runx perturbations were introduced. g, Histograms display the aggregated enrichment scores of indicated pathways (ETP, DN2, DN3, and Myeloid pathways) in each cell computed from curated reference gene sets by ssGSEA. Cells were grouped by types of Runx perturbation and timepoints.

Journal: bioRxiv

Article Title: Runx factors launch T-cell and innate lymphoid programs via direct and gene network-based mechanisms

doi: 10.1101/2022.11.18.517146

Figure Lengend Snippet: a, Experimental schematics for single cell RNA-seq (scRNA-seq) are depicted. Each experimental condition group was marked by a different hashtag oligo (HTO) and equal numbers of cells from each were pooled for scRNA-seq. Two independent scRNA-seq experiments were performed. See Methods for details. b-d, UMAP2-3 illustrate scRNA- seq data from Runx1 OE and Runx1/Runx3 double knockout (KO) in Phase 1. b, Color intensity in UMAP displays expression levels of indicated genes, which are informative to represent different T-development stages. c, Types of Runx perturbation (control, Runx1 OE, and Runx1/Runx3 KO cells) with cells from different experimental time points are highlighted in UMAP2-3 space. d, Cells from scRNA-seq are colored by Louvain clusters. e, Area-proportional Venn diagram shows the number of Runx-activated (blue) and - inhibited genes (orange) in OE and/or KO perturbations. The common, core target gene numbers are shown in red. f, Scatter plots compare Log 2 fold-changes of Runx target gene expression in Runx1 OE and Runx KO conditions at d2-d3 (left) or d4-d6 (right) after Runx perturbations were introduced. g, Histograms display the aggregated enrichment scores of indicated pathways (ETP, DN2, DN3, and Myeloid pathways) in each cell computed from curated reference gene sets by ssGSEA. Cells were grouped by types of Runx perturbation and timepoints.

Article Snippet: Three gRNAs were utilized to target each Runx paralog (Addgene #189799, #189800, #189801, #189802, #189803, #189804, #189805, #189806).

Techniques: RNA Sequencing, Double Knockout, Expressing, Control, Targeted Gene Expression

a, Heatmap represents Runx1 or Runx3 DNA binding patterns in non-promoter regions from indicated cells. Orange tracks were derived from experimental cells and blue tracks were obtained from unperturbed Phase 1 ( in vitro DN1) and Phase 2 (thymic DN3) pro-T cells (two independent C&R experiments for each condition). Stage-dependent chromatin accessibility patterns in normal cells at Group 2a, Group 2b, and Group 4 sites are shown on the right with percent of total peaks in a group. b, Representative UCSC genome browser tracks display Runx binding (C&R) together with published HiC PC1 values, chromatin accessibility (ATAC) profiles, and binding sites of loop forming machinery (CTCF and SMC). Enhancer regions near Bcl11b , Ets1 , and Zbtb16 are displayed. c, Runx motif frequencies in different Groups of Runx binding sites are illustrated as density plots. d, Violin plot demonstrates the best Runx motif score distribution in each Groups of Runx binding sites. Two sample KS test (comparing to Group3 motif scores). *** p<0.001. The horizontal dotted line shows the threshold PWM score to be considered to harbor the Runx motif. Thin vertical black lines mark 1.5x interquartile range and thick vertical black lines show interquartile range. The red lines with white circles indicate median values. e, Testing hypothesis that Runx1 OE accesses sites conditionally occupied in other pro-T related contexts. Area-proportional Venn diagrams show analysis strategy to identify Runx binding sites appearing specifically in Bcl11b knockout DN2b/DN3 cells (left), and ILC2-specific Runx binding sites (middle; Runx1, right; Runx3). f, Bar graph shows percentages of Group 4 peaks overlapping with indicated Runx binding site types.

Journal: bioRxiv

Article Title: Runx factors launch T-cell and innate lymphoid programs via direct and gene network-based mechanisms

doi: 10.1101/2022.11.18.517146

Figure Lengend Snippet: a, Heatmap represents Runx1 or Runx3 DNA binding patterns in non-promoter regions from indicated cells. Orange tracks were derived from experimental cells and blue tracks were obtained from unperturbed Phase 1 ( in vitro DN1) and Phase 2 (thymic DN3) pro-T cells (two independent C&R experiments for each condition). Stage-dependent chromatin accessibility patterns in normal cells at Group 2a, Group 2b, and Group 4 sites are shown on the right with percent of total peaks in a group. b, Representative UCSC genome browser tracks display Runx binding (C&R) together with published HiC PC1 values, chromatin accessibility (ATAC) profiles, and binding sites of loop forming machinery (CTCF and SMC). Enhancer regions near Bcl11b , Ets1 , and Zbtb16 are displayed. c, Runx motif frequencies in different Groups of Runx binding sites are illustrated as density plots. d, Violin plot demonstrates the best Runx motif score distribution in each Groups of Runx binding sites. Two sample KS test (comparing to Group3 motif scores). *** p<0.001. The horizontal dotted line shows the threshold PWM score to be considered to harbor the Runx motif. Thin vertical black lines mark 1.5x interquartile range and thick vertical black lines show interquartile range. The red lines with white circles indicate median values. e, Testing hypothesis that Runx1 OE accesses sites conditionally occupied in other pro-T related contexts. Area-proportional Venn diagrams show analysis strategy to identify Runx binding sites appearing specifically in Bcl11b knockout DN2b/DN3 cells (left), and ILC2-specific Runx binding sites (middle; Runx1, right; Runx3). f, Bar graph shows percentages of Group 4 peaks overlapping with indicated Runx binding site types.

Article Snippet: Three gRNAs were utilized to target each Runx paralog (Addgene #189799, #189800, #189801, #189802, #189803, #189804, #189805, #189806).

Techniques: Binding Assay, Derivative Assay, In Vitro, Knock-Out

a, Density plots illustrate motif frequencies for PU.1, TCF1 (Tcf7), bHLH, and GATA factors in different types of Runx binding sites. b, Runx1, Runx3 (blue), PU1 (purple) , TCF1 (red), E2A and HEB (green) binding profiles in non-promoter regions under unperturbed Phase 1 or Phase 2 conditions are shown. Runx1 binding patterns in empty vector control and Runx1 OE transduced conditions are displayed in orange tracks (left). Stage-preferential dynamic binding groups are indicated as color bars. Group 1, Phase 1-preferential; Group 2a, Phase 2-preferential and precociously occupied by OE; Group 2b, Phase 2-preferential but not occupied by OE; Group 3, Phase 1 & Phase 2 shared; Group 4a, OE-specific and open sites; Group 4b, OE-specific and closed sites. The numbers on the right side indicate percent of each group of peaks within the same color bar. TCF1, E2A, and HEB binding sites were measured in independent replicates using C&R from thymic DN3 cells. PU.1 occupancy was previously determined using ChIP-seq . c, Number of Runx binding sites co-occupied with PU.1 or TCF1 or E-proteins were enumerated and their percentages in each group are shown using a bar graph. d, Representative UCSC genome browser tracks near Gata3 and Myb show indicated TF binding profiles. e, shRNA against Tcf7 or random control shRNA was introduced to bone-marrow progenitor cells in combination with Runx1-OE or empty control vector, then the progenitor cells were co-cultured with OP9-Dll1 for 2 days. Bar graph summarizes Bcl11b-mCitrine and CD25 expression levels measured by flow cytometry with mean and standard deviation. n=4 independent experiments, Two-way ANOVA. ***=p-value<0.001, **=p-value<0.01, ns=not significant.

Journal: bioRxiv

Article Title: Runx factors launch T-cell and innate lymphoid programs via direct and gene network-based mechanisms

doi: 10.1101/2022.11.18.517146

Figure Lengend Snippet: a, Density plots illustrate motif frequencies for PU.1, TCF1 (Tcf7), bHLH, and GATA factors in different types of Runx binding sites. b, Runx1, Runx3 (blue), PU1 (purple) , TCF1 (red), E2A and HEB (green) binding profiles in non-promoter regions under unperturbed Phase 1 or Phase 2 conditions are shown. Runx1 binding patterns in empty vector control and Runx1 OE transduced conditions are displayed in orange tracks (left). Stage-preferential dynamic binding groups are indicated as color bars. Group 1, Phase 1-preferential; Group 2a, Phase 2-preferential and precociously occupied by OE; Group 2b, Phase 2-preferential but not occupied by OE; Group 3, Phase 1 & Phase 2 shared; Group 4a, OE-specific and open sites; Group 4b, OE-specific and closed sites. The numbers on the right side indicate percent of each group of peaks within the same color bar. TCF1, E2A, and HEB binding sites were measured in independent replicates using C&R from thymic DN3 cells. PU.1 occupancy was previously determined using ChIP-seq . c, Number of Runx binding sites co-occupied with PU.1 or TCF1 or E-proteins were enumerated and their percentages in each group are shown using a bar graph. d, Representative UCSC genome browser tracks near Gata3 and Myb show indicated TF binding profiles. e, shRNA against Tcf7 or random control shRNA was introduced to bone-marrow progenitor cells in combination with Runx1-OE or empty control vector, then the progenitor cells were co-cultured with OP9-Dll1 for 2 days. Bar graph summarizes Bcl11b-mCitrine and CD25 expression levels measured by flow cytometry with mean and standard deviation. n=4 independent experiments, Two-way ANOVA. ***=p-value<0.001, **=p-value<0.01, ns=not significant.

Article Snippet: Three gRNAs were utilized to target each Runx paralog (Addgene #189799, #189800, #189801, #189802, #189803, #189804, #189805, #189806).

Techniques: Binding Assay, Plasmid Preparation, Control, ChIP-sequencing, shRNA, Cell Culture, Expressing, Flow Cytometry, Standard Deviation

a, Gene regulatory network analysis strategy is shown. Cells were grouped by Runx perturbation condition and pseudotime category to compute predicted target gene activity using SCENIC (pySCENIC, see Methods). b-c, SCENIC-predicted regulon activities for indicated TFs are represented as a heatmap (b) or highlighted on the UMAP2/UMAP3 manifold (c). The expressed regulons scoring adjusted p-value < 1e-10 from at least two different pairwise comparisons using Kolmogorov–Smirnov tests were selected to visualize. d, The members of each regulon were overlapped with Runx DEGs defined by KO and/or OE from . Then the numbers of overlapping predicted input regulons were enumerated per functionally responding Runx target gene or per non-DEG, and the results displayed as cumulative density functions. Kolmogorov–Smirnov test p-values were calculated by comparing Runx-activated or Runx-repressed DEGs with non-DEGs. Activated genes’ p-value=1.55e-15, inhibited genes’ p-value=8.88e-16. e, Curated Runx DEGs regulon memberships predicting input relationships are displayed as matrices. Colored cells in matrix indicate that a given Runx DEG (rows) is a member of a given regulon (columns). Blue; Runx-activated genes, orange; Runx-inhibited genes. f, Area-proportional Venn diagrams display overlap between functionally responsive Runx target genes with previously determined functional target genes of PU.1 , GATA3 , TCF1 , and Bcl11b .

Journal: bioRxiv

Article Title: Runx factors launch T-cell and innate lymphoid programs via direct and gene network-based mechanisms

doi: 10.1101/2022.11.18.517146

Figure Lengend Snippet: a, Gene regulatory network analysis strategy is shown. Cells were grouped by Runx perturbation condition and pseudotime category to compute predicted target gene activity using SCENIC (pySCENIC, see Methods). b-c, SCENIC-predicted regulon activities for indicated TFs are represented as a heatmap (b) or highlighted on the UMAP2/UMAP3 manifold (c). The expressed regulons scoring adjusted p-value < 1e-10 from at least two different pairwise comparisons using Kolmogorov–Smirnov tests were selected to visualize. d, The members of each regulon were overlapped with Runx DEGs defined by KO and/or OE from . Then the numbers of overlapping predicted input regulons were enumerated per functionally responding Runx target gene or per non-DEG, and the results displayed as cumulative density functions. Kolmogorov–Smirnov test p-values were calculated by comparing Runx-activated or Runx-repressed DEGs with non-DEGs. Activated genes’ p-value=1.55e-15, inhibited genes’ p-value=8.88e-16. e, Curated Runx DEGs regulon memberships predicting input relationships are displayed as matrices. Colored cells in matrix indicate that a given Runx DEG (rows) is a member of a given regulon (columns). Blue; Runx-activated genes, orange; Runx-inhibited genes. f, Area-proportional Venn diagrams display overlap between functionally responsive Runx target genes with previously determined functional target genes of PU.1 , GATA3 , TCF1 , and Bcl11b .

Article Snippet: Three gRNAs were utilized to target each Runx paralog (Addgene #189799, #189800, #189801, #189802, #189803, #189804, #189805, #189806).

Techniques: Activity Assay, Functional Assay