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

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

Journal: Pharmaceuticals

Article Title: GL-V9 Promotes Autophagy-Mediated YAP1 Degradation and Activates Mitochondrial Apoptosis in PDAC Cells

doi: 10.3390/ph17101352

Figure Lengend Snippet: GL-V9 induces apoptosis through decreasing the expression of YAP1 in PDAC cells. ( A ) Analysis of the TCGA database revealed significant variation in YAP1 expression across different cancer types; ( B ) the expression of YAP1 in pancreatic cancer clinical samples based on the TCGA database (** p < 0.01); ( C ) the effect of YAP1 expression on pancreatic cancer patients’ survival; ( D ) Western blot analysis of YAP1 expression in HPNE, BXPC-3, PANC-1, MiaPaCa-2, and CaPan-2 cells (ns: no significance, *** p < 0.001); ( E ) Western blot analysis of YAP1 and P-YAP1 after BXPC-3 and PANC-1 cells were treated with GL-V9 (0, 10, 20, or 30 μM) for 24 h (ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001); ( F ) the effect of GL-V9 on nuclear localization of YAP1 (scale bar, 25 μm); ( G ) the effect of GL-V9 on YAP1 mRNA level in pancreatic cancer cells (ns: no significance).

Article Snippet: The primary antibodies used in Western blotting included ACTB, Bax, Bcl-2, Bcl-xL, caspase 3, caspase 8, caspase 9, Cyt C, GAPDH, Mcl-1, mTOR, PARP-1, P-YAP1, VDAC1, YAP1 and β-tubblin (1:1000, ABclonal, Wuhan, China), ATG5 and BECN1 (1:1000, Bioswap, Hangzhou, China), TEAD1 (1:1000, FineTest, Wuhan, China), Ubiquitin (1:1000, Immunoway, Plano, TX, USA) AKT, Gal-3, LAMP1, LAMP2, LC3, p62, P-AKT, and TFEB (1:1000, Proteintech, Wuhan, China).

Techniques: Expressing, Western Blot

Journal: Pharmaceuticals

Article Title: GL-V9 Promotes Autophagy-Mediated YAP1 Degradation and Activates Mitochondrial Apoptosis in PDAC Cells

doi: 10.3390/ph17101352

Figure Lengend Snippet: GL-V9 induces degradation of YAP1 through the autophagy–lysosome pathway. ( A ) The impact of GL-V9 on the stability of YAP1 protein was investigated. BXPC-3 and PANC-1 cells were treated with 10 μg/mL CHX and 30 μM GL-V9 for 0, 6, 12, and 24 h (* p < 0.05, *** p < 0.001); ( B ) the effect of Baf A1 on YAP1 protein by GL-V9. BXPC-3 and PANC-1 cells were pre-treated with 50 nM for 2 h and then co-treated with 30 μM GL-V9 for 24 h (*** p < 0.001); ( C ) the effect of GL-V9 on the location of YAP1 and LAMP2, (scale bar, 25 μm); ( D ) the effect of GL-V9 on the location of YAP1 and p62, (scale bar, 25 μm); ( E ) the effect of the binding of YAP1 with ubiquitin, p62, and TEAD1 induced by GL-V9; ( F ) the expression of YAP1 and Bcl-2 in YAP1 overexpression BXPC-3 cells (ns: no significance, ** p < 0.01, *** p < 0.001); ( G ) YAP1 overexpression reversed the level of apoptosis protein induced by GL-V9 (ns: no significance, *** p < 0.001).

Article Snippet: The primary antibodies used in Western blotting included ACTB, Bax, Bcl-2, Bcl-xL, caspase 3, caspase 8, caspase 9, Cyt C, GAPDH, Mcl-1, mTOR, PARP-1, P-YAP1, VDAC1, YAP1 and β-tubblin (1:1000, ABclonal, Wuhan, China), ATG5 and BECN1 (1:1000, Bioswap, Hangzhou, China), TEAD1 (1:1000, FineTest, Wuhan, China), Ubiquitin (1:1000, Immunoway, Plano, TX, USA) AKT, Gal-3, LAMP1, LAMP2, LC3, p62, P-AKT, and TFEB (1:1000, Proteintech, Wuhan, China).

Techniques: Binding Assay, Ubiquitin Proteomics, Expressing, Over Expression

Journal: Pharmaceuticals

Article Title: GL-V9 Promotes Autophagy-Mediated YAP1 Degradation and Activates Mitochondrial Apoptosis in PDAC Cells

doi: 10.3390/ph17101352

Figure Lengend Snippet: GL-V9 inhibits the growth of PDAC in vivo via activating autophagy and inducing apoptosis. ( A ) The weight of tumors for six groups of animals were compared. Data represent mean ± SD. *** p < 0.001; ( B ) the tumor volume was measured once every three days; ( C ) the body weight of mice was measured once every three days within 27 days of administration; ( D ) Western blot analysis of Bax, Bcl-2, Bcl-xL, pro-caspase 3, pro-caspase 9, cleaved caspase 9, and cleaved caspase 3 in tumor xenograft tissues. ACTB was used as loading control (ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001); ( E ) the expression of apoptosis-related proteins in tumor xenograft tissues were measured by immunohistochemistry. Scale bars, 50 μm; ( F ) Western blot analysis of ATG5, LAMP1, BECN1, and YAP1 in tumor xenograft tissues. ACTB was used as loading control (ns: no significance, * p < 0.05, ** p < 0.01, *** p < 0.001); ( G ) the expression of autophagy-related proteins in tumor xenograft tissues was measured by immunohistochemistry. Scale bars, 50 μm.

Article Snippet: The primary antibodies used in Western blotting included ACTB, Bax, Bcl-2, Bcl-xL, caspase 3, caspase 8, caspase 9, Cyt C, GAPDH, Mcl-1, mTOR, PARP-1, P-YAP1, VDAC1, YAP1 and β-tubblin (1:1000, ABclonal, Wuhan, China), ATG5 and BECN1 (1:1000, Bioswap, Hangzhou, China), TEAD1 (1:1000, FineTest, Wuhan, China), Ubiquitin (1:1000, Immunoway, Plano, TX, USA) AKT, Gal-3, LAMP1, LAMP2, LC3, p62, P-AKT, and TFEB (1:1000, Proteintech, Wuhan, China).

Techniques: In Vivo, Western Blot, Control, Expressing, Immunohistochemistry

Journal: Journal of Nanobiotechnology

Article Title: Hyaluronic acid-based reduction responsive nanoparticles for Improved anti-psoriasis effects of traditional Chinese herb monomer oleanolic acid via blocking YAP-AREG axis

doi: 10.1186/s12951-026-04264-x

Figure Lengend Snippet: NPH modulates the YAP signaling pathway to exert anti-psoriatic effects. ( A ) IHC staining images of p-YAP1, active YAP1, and YAP1 in psoriatic lesions from different groups and ( B ) quantitative analysis of the average positive intensity of p-YAP1, active YAP1, and YAP1 in the epidermal layer of the lesions. Bar = 100 μm and 20 μm. ( C ) IF staining images of p-YAP1 and YAP1 in psoriatic lesions from different groups. Bar = 100 μm and 25 μm, and ( D ) the ratios of p-YAP1/YAP1. ( E ) Western blot analysis of Hippo/YAP pathway components in psoriatic lesions from different groups. ( F ) The ratios of p-YAP1/YAP1, p-LATS1/LATS1, and p-MST1/MST1 quantified from Fig. 8E ( n = 3). ( G ) Expression of YAP1 and YAP1-regulated genes in the psoriatic lesions from different groups by RT-qPCR. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. = no significance

Article Snippet: Skin tissues from the Normal, IMQ-PBS, and IMQ-NPH groups were subjected to double immunofluorescence staining for p-YAP1 (Abmart, T55743 , 1:1000 dilution) and YAP1 (Abmart, PAQ6610, 1:250 dilution).

Techniques: Immunohistochemistry, Staining, Western Blot, Expressing, Quantitative RT-PCR