Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: YAP1 is upregulated in HBZY-1 cells treated with high glucose. (A) RT-qPCR was used to measure the expression of YAP1 in HBZY-1 cells treated with 30 mmol/l serum-free DMEM medium for 0, 6, 12, 24 and 48 h, respectively. (B) Western blotting was used to measure the expression of YAP1 in HBZY-1 cells treated with 30 mmol/l serum-free DMEM medium for 0, 6, 12, 24 and 48 h, respectively. (C) Transfection efficiency of YAP1 small interfering RNA was detected by RT-qPCR. *** P<0.001 vs. HG 0 h; ### P<0.001 vs. HG + si-NC. YAP1, Yes-associated protein 1; RT-qPCR, reverse transcription-quantitative PCR; si-, small interfering RNA; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Small Interfering RNA, Real-time Polymerase Chain Reaction, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: Knockdown of YAP1 inhibited the proliferation of HBZY-1 cells induced by high glucose. (A) Effect of YAP1 knockdown on the viability of HBZY-1 cells treated with high glucose was detected by Cell Counting Kit-8 assay. (B) Effect of YAP1 knockdown on the expression of PCNA in the HBZY-1 cells treated with high glucose was measured by western blotting. * P<0.05 vs. HG + si-NC. YAP1, Yes-associated protein 1; PCNA, proliferating cell nuclear antigen; si-, small interfering RNA; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Cell Counting, Expressing, Western Blot, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: Knockdown of YAP1 suppresses the inflammation of HBZY-1 cells induced by high glucose. (A) Effect of YAP1 knockdown on the expressions of pro-inflammatory factors (TNF-α, IL-1β and IL-6) was detected by enzyme-linked immunosorbent assay. (B) Effect of YAP1 knockdown on the expression of MCP-1 was detected by reverse transcription-quantitative PCR. (C) Effect of YAP1 knockdown on the expression of MCP-1 was detected by western blotting. ** P<0.01 and *** P<0.001 vs. HG + si-NC. YAP1, Yes-associated protein 1; si-, small interfering RNA; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol; TNF, tumor necrosis factor; IL, interleukin; MCP-1, monocyte chemoattractant protein-1.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: Knockdown of YAP1 inhibits the deposition of extracellular matrix and fibrosis in HBZY-1 cells induced by high glucose. (A) Effect of YAP1 knockdown on the expressions of collagen IV, α-SMA and FN was detected by western blotting. (B and C) Effect of YAP1 knockdown on the expressions of collagen IV was detected by immunofluorescence. Scale bars, 50 µm. (D) Effect of YAP1 knockdown on the expressions of fibrogenic factors CTGF and TGF-β1 was detected by western blotting. * P<0.05, ** P<0.01 and *** P<0.001 vs. HG + si-NC. YAP1, Yes-associated protein 1; si-, small interfering RNA; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol; SMA, smooth muscle actin; FN, fibronectin; CTGF, connective tissue growth factor; TGF, transforming growth factor.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Western Blot, Immunofluorescence, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: Knockdown of YAP1 inhibited NF-κB/JMJD3 signal pathway in HBZY-1 cells induced by high glucose. (A) Effect of YAP1 knockdown on the phosphorylation levels of IκBα and p65 were detected by western blotting. (B) Effect of YAP1 knockdown on the expression of JMJD3 was detected by western blotting. * P<0.05, ** P<0.01 and *** P<0.001 vs. HG + si-NC. YAP1, Yes-associated protein 1; si-, small interfering RNA; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol; NF-κB, nuclear factor-κB; JMJD3, Jumonji domain-containing protein D3; p-, phosphorylated-; IκBα, inhibitor of NF-κB.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Western Blot, Expressing, Small Interfering RNA, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: Inhibitors of NF-κB/JMJD3 signal pathway attenuates the facilitatory effects of YAP1 on the proliferation and inflammation of HBZY-1 cells induced by high glucose. (A) The efficiency of YAP1 overexpression was determined by RT-qPCR. (B) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression levels of IκBα and p65 were detected by western blotting. (C) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the viability of HBZY-1 cells treated with high glucose was detected by Cell Counting Kit-8 assay. (D) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression of PCNA in the HBZY-1 cells treated with high glucose was measured by western blotting. (E) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expressions of pro-inflammatory factors (TNF-α, IL-1β and IL-6) was detected by enzyme-linked immunosorbent assay. (F) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression of MCP-1 was detected by RT-qPCR. (G) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression of MCP-1 was detected by western blotting. ** P<0.01 vs. HG + Oe-NC. # P<0.05, ## P<0.01 and ### P<0.001 vs. HG + Oe-YAP1. YAP1, Yes-associated protein 1; RT-qPCR, reverse transcription-quantitative PCR; Oe, overexpression vector; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol; NF-κB, nuclear factor-κB; JMJD3, Jumonji domain-containing protein D3; p-, phosphorylated-; IκBα, inhibitor of NF-κB; TNF, tumor necrosis factor; IL, interleukin; MCP-1, monocyte chemoattractant protein-1.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Cell Counting, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: YAP1 promotes high glucose-induced inflammation and extracellular matrix deposition in glomerular mesangial cells by modulating NF-κB/JMJD3 pathway

doi: 10.3892/etm.2021.10784

Figure Lengend Snippet: Inhibitors of NF-κB/JMJD3 signal pathway attenuates the facilitatory effects of YAP1 on the deposition of extracellular matrix and fibrosis of HBZY-1 cells induced by high glucose. (A) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression levels of collagen IV, α-SMA and FN was detected by western blotting. (B and C) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression levels of collagen IV was detected by immunofluorescence. Scale bars, 50 µm. (D) Effect of the inhibitors of NF-κB/JMJD3 signal pathway on the expression levels of fibrogenic factors CTGF and TGF-β1 was detected by western blotting. # P<0.05 and ## P<0.01 vs. HG + Oe-YAP1. YAP1, Yes-associated protein 1; Oe, overexpression vector; NC, negative control; HG, high-glucose group; NG, normal control group; MA, mannitol; NF-κB, nuclear factor-κB; JMJD3, Jumonji domain-containing protein D3; SMA, smooth muscle actin; FN, fibronectin; CTGF, connective tissue growth factor; TGF, transforming growth factor.

Article Snippet: The YAP1 overexpression vector pcDNA-TRIM31 (Oe-YAP1) and empty control vector pcDNA (Oe-NC) were constructed by Shanghai GenePharma Co., Ltd. Small interfering RNA (siRNA) against YAP1 (si-YAP1) and siRNA negative control (si-NC) were synthesized by Shanghai GenePharma Co., Ltd. A final concentration of 100 nM plasmids were transfected into cells using Lipofectamine ® 2000 (Thermo Fisher Scientific, Inc.) transfection reagent.

Techniques: Expressing, Western Blot, Immunofluorescence, Over Expression, Plasmid Preparation, Negative Control

Journal: The FASEB Journal

Article Title: Sirtuin 6 protects against hepatic fibrogenesis by suppressing the YAP and TAZ function

doi: 10.1096/fj.202200522r

Figure Lengend Snippet: FIGURE 9 SIRT6 deacetylates YAP1 at multiple lysine residues. (A) A diagram of human YAP1 domain structure and several known acetylated lysine residues. (B) Real-time PCR analysis of CYR61, CTGF, and ANKRD1 mRNAs in the LX-2 cells transfected with either WT or mutant human YAP1 plasmids in the presence of 5 ng/ml TGF-β1 (n = 3). (C) YAP1 acetylation analysis in LX-2 cells transfected with WT or mutant human YAP1 plasmids together with vector or SIRT6 plasmids in the presence of 5 ng/ml TGF-β1. Data are presented as mean ± SEM. *p < .05, **p < .01, ***p < .001 versus vector control, and #p < .05, ##p < .001, ###p < .001 versus WT YAP.

Article Snippet: Human YAP1 was a gift from Yosef Shaul (Addgene, Cambridge, MA; plasmid # 18881; http:// n2t.net/addge ne:18881; RRID:Addgene_18881).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Mutagenesis, Plasmid Preparation, Control

Journal: Scientific Reports

Article Title: Zebrafish mutants and TEAD reporters reveal essential functions for Yap and Taz in posterior cardinal vein development

doi: 10.1038/s41598-018-27657-x

Figure Lengend Snippet: Hsa.CTGF -based Yap1/Taz zebrafish reporter line. ( A ) ctgf promoter contains three Yap1/Taz responsive elements (RE) and is remarkably conserved between zebrafish and humans. The promoter region upstream of the 5′UTR (uppercase) of the zebrafish ctgfa and human CTGF genes contain each three TEAD DNA-binding sites (YAP1/TAZ REs; in light blue) just before the TATA box (in yellow). Their sequences, orientation and distances are remarkably conserved between zebrafish and humans. ( B ) Schematic representation of the Hsa . CTGF -based Yap1/Taz reporter construct. The Yap1/Taz-responsive fragment of the construct is derived from the human CTGF promoter. It contains the pathway-specific REs MCAT and the TATA box, and regulates the expression of the downstream reporter gene. ( C ) Overview of a Tg(Hsa . CTGF:nlsmCherry) larva.

Article Snippet: Anti-YAP1 (63.7) monoclonal antibody (sc-101199) was from Santa Cruz, anti-TAZ (560235) monoclonal antibody was from BD Bioscience.

Techniques: Binding Assay, Construct, Derivative Assay, Expressing

Journal: Scientific Reports

Article Title: Zebrafish mutants and TEAD reporters reveal essential functions for Yap and Taz in posterior cardinal vein development

doi: 10.1038/s41598-018-27657-x

Figure Lengend Snippet: Hsa.CTGF -based zebrafish transgenic lines are bona fide Yap1/Taz reporter. ( A ) Morpholino-mediated Yap1/Taz knockdown reduces Hsa . CTGF reporter signal (in red). Two splice morpholinos, targeting respectively Yap1 and Taz pre-mRNAs, were co-injected in one-cell stage Tg(Hsa . CTGF:nlsmCherry) ia49 embryos. ( B – D ) Constitutive activation of Yap1/Taz-mediated transcription increases Hsa . CTGF reporter signal (in red). The mRNA coding for constitutively active versions of YAP1 ( B ), TAZ ( C ) or TEAD ( D ) were injected in one-cell stage Tg(Hsa . CTGF:nlsmCherry) ia49 embryos. The fluorescent reporter expression was documented and quantified at 24 hpf. Control morpholino-injected: n = 20; Yap/Taz morpholino-injected: n = 29; controls for YAP-5SA: n = 12; YAP-5SA mRNA-injected: n = 12; controls for TAZ-4SA: n = 57; TAZ-4SA mRNA-injected: n = 56; controls for TEAD-VP16: n = 20; TEAD-VP16 mRNA-injected: n = 20. A.U.: arbitrary units; *p < 0.05; ***p < 0.001.

Article Snippet: Anti-YAP1 (63.7) monoclonal antibody (sc-101199) was from Santa Cruz, anti-TAZ (560235) monoclonal antibody was from BD Bioscience.

Techniques: Transgenic Assay, Knockdown, Injection, Activation Assay, Expressing, Control

Journal: Scientific Reports

Article Title: Zebrafish mutants and TEAD reporters reveal essential functions for Yap and Taz in posterior cardinal vein development

doi: 10.1038/s41598-018-27657-x

Figure Lengend Snippet: Tg(Hsa . CTGF:nlsmCherry) ia49 reporter activity is prominent in the endothelium. ( A – D ) Reporter expression in the endothelium during development from 20 to 48 hpf. ( E – I ) Reporter expression in the adult endothelium. ( A ) Confocal sagittal section of the trunk of a 20 hpf Tg(Hsa . CTGF:nlsmCherry) ia49 /Tg(kdrl:GFP) double transgenic embryo. Tg(kdrl:GFP) expresses GFP in all endothelial cells. Yap1/Taz are active in the endothelial cells in the developing vessels, as shown by the co-localization between the two signals. ( B – D ) Confocal Z-stack projections of the head region (side view in ( B ), dorsal view in ( D )) and the trunk ( C ) at 48 hpf. Reporter signal co-localizes with kdrl :GFP expression throughout the embryo. ( E , F ) Confocal Z-stack projection of brain ( E ) and liver ( F ) tissue of a double transgenic adult fish, displaying Yap1/Taz reporter activation in the endothelium of respectively the cerebral and hepatic vascular networks. The insets represent zoomed views highlighting the co-localization between Hsa . CTGF :nlsmCherry and kdrl :GFP. ( G – I ) Fluorescent microscope images of adult caudal fin in Tg(kdrl:GFP) ( G ) and Tg(Hsa . CTGF:nlsmCherry) ia49 ( H ). Lateral views, anterior to the left, dorsal to the top. Arterial (white arrows) and venous (black arrows) bloodstream is indicated. Yap1/Taz reporter activity is stronger in the veins running laterally to the bony fin rays with respect to arteries inside the bony rays, as highlighted in the single channels and merge magnifications ( I –I”’). a: artery; v-vein. Scale bar: 100 μm.

Article Snippet: Anti-YAP1 (63.7) monoclonal antibody (sc-101199) was from Santa Cruz, anti-TAZ (560235) monoclonal antibody was from BD Bioscience.

Techniques: Activity Assay, Expressing, Transgenic Assay, Activation Assay, Microscopy

Journal: Scientific Reports

Article Title: Zebrafish mutants and TEAD reporters reveal essential functions for Yap and Taz in posterior cardinal vein development

doi: 10.1038/s41598-018-27657-x

Figure Lengend Snippet: Yap1/Taz are required for PCV development. ( A ) Gross morphology of 20 hpf WT and yap1 −/− ;taz + / − embryos. Red arrows point to the undulating notochord. ( A’ ) Gross morphology of 32 hpf WT and yap1 −/− ;taz + / − embryos. ( B , B’ ) Transverse sections of the trunk revealing the relative positions of the neural tube (NT), dorsal aorta (DA) and posterior cardinal vein (PCV). The PCV of yap1 −/− ;taz + / − animals has deviated from the midline and appears to split into two lumenized vessels (arrows). White signal, etv2: EGFP transgene expression; red signal, lyve1b :DsRed transgene expression. The PVC lumen is marked with an asterisk (*), while the DA lumen is marked with a yellow “ + ”. ( C , D ) Analysis of the deviation of the PCV from the midline in 48 hpf embryos. The data are obtained from a series of transverse sections starting from the caudal end of the yolk tube and moving 50 sections rostrally. The NT, DA and PCV were manually demarcated and the deviation of the PCV from the midline was defined as β = 180°-ϴ. With ϴ (see panel B ) we defined the angle formed by the NT, DA, and PCV in each transverse section. Each value of β was plotted for WT and yap1 −/− ;taz + / − trunks at 48 hpf. Each line represents a single animal. 4/6 of yap1 −/− ;taz + / − embryos exhibited a PCV that appears to split into two lumenized vessels that are lyve1b :DsRed positive (marked with a circle on the graph). Red horizontal lines above and below 0° are the WT maxima and minima. P values were calculated by the F-test, which tests whether the spread of angles (180° - ϴ) between WT and mutants is the same. σ mut 2 : variance of (180° - ϴ) in yap1 −/− ;taz + / − animals; σ WT 2 : variance of (180° - ϴ) in WT animals. ( E ) Whole mount in situ hybridization (WISH) for the expression of efnb2a and mrc1a , markers of arteries and veins, respectively. Red arrows point to expression of mrc1a in the region of the DA. The fraction of the embryos exhibiting the phenotype shown in each image was reported in the upper right corner of the corresponding panel. Scale bars, 100 μm.

Article Snippet: Anti-YAP1 (63.7) monoclonal antibody (sc-101199) was from Santa Cruz, anti-TAZ (560235) monoclonal antibody was from BD Bioscience.

Techniques: Expressing, In Situ Hybridization

Journal: Scientific Reports

Article Title: Zebrafish mutants and TEAD reporters reveal essential functions for Yap and Taz in posterior cardinal vein development

doi: 10.1038/s41598-018-27657-x

Figure Lengend Snippet: Yap1/Taz activity upregulation promotes vessel sprouting. ( A – C ) Confocal Z-stack projections of the midtrunk region of Tg(kdrl:GFP) 32 hpf embryos. ( A ) Representative image of a control injected embryo. ( B , B’ ) Two TAZ-4SA mRNA injected embryos, showing an aberrant ISV sprout (arrowhead in B ) and an anastomosis between adjacent ISVs (arrowhead in B’ ). ( C ) A mosaic embryo injected with pDestTol2CG2-fli1a-TAZ-4SA plasmid, showing three anastomosis between adjacent ISVs (arrowheads in C ). ( D , E ) Quantification of the aberrant sprouting caused by Yap1/Taz activity upregulation. The number of non-anastomosed aberrant sprouts ( D ) and the number of anastomosis ( E ) between adjacent ISVs were evaluated. Both phenomena, observed in TAZ-4SA mRNA and pDestTol2CG2-fli1a-TAZ-4SA mosaic injected embryos, are extremely rare or absent at all in the controls. Controls: n = 49; TAZ-4SA mRNA-injected: n = 42; TEAD-VP16 mRNA-injected: n = 31; pDestTol2CG2-fli1a-TAZ-4SA plasmid-injected: n = 16. ( F , F’ ) Tg(Hsa . CTGF:nlsmCherry) ia49 /Tg(kdrl:GFP) double transgenic embryos injected with the pDestTol2CG2-fli1a:TAZ-4SA vector. A strong overactivation of the Hsa . CTGF reporter signal was observed in the nuclei of the endothelial cells undergoing anomalous sprouting (arrowheads in F ) with respect to the other normal ISVs. ( G ) In mosaic embryos injected with the PCS2-fli1a:CAYAP-mKate plasmid a specific expression of the mKate was reported in conjunction with the anomalous endothelial sprouts (arrowhead). The plasmid is endothelium-specific, as highlighted by the co-localization (arrows) between the mosaic mKate and the GFP of the stable transgenic line Tg(kdrl:EGFP) . Lateral view, anterior to the left, dorsal to the top. ***p < 0.001. ISV: intersegmental vessel; DLAV: dorsal longitudinal anastomotic vessel. Scale bar: 50 µm.

Article Snippet: Anti-YAP1 (63.7) monoclonal antibody (sc-101199) was from Santa Cruz, anti-TAZ (560235) monoclonal antibody was from BD Bioscience.

Techniques: Activity Assay, Control, Injection, Plasmid Preparation, Transgenic Assay, Expressing