Journal: bioRxiv

Article Title: UDP-glucose pyrophosphorylase 2, a regulator of glycosylation and glycogen, is essential for pancreatic cancer growth

doi: 10.1101/2020.10.13.337998

Figure Lengend Snippet: A, Scoring of tissue microarrays containing 78 pancreatic ductal adenocarcinoma samples immunohistochemically stained for YAP or UGP2. +, ++, +++ represent combined scoring of automated area x intensity measurements. **** p < 0.0001 by Chi-square test. B, qPCR for UGP2 messenger RNA in Panc1, MiaPaca2, and Suit2 cells with shYAP or empty vector. *** p < 0.001. C, Immunoblot on lysates from Panc1, MiaPaca2, and Suit2 cells stably expressing shYAP or empty vector and probed as indicated. D-E, Immunoblots on lysates from MCF10A (D) and 293T (E) cells stably expressing wild-type YAP, YAP5SA, YAP S94A, or empty vector, probed as indicated. F, ChIP-seq for YAP1 and TEAD4 at the UGP2 locus. G, Targeted PCR using two sets of primers (p1 and p2) in the UGP2 promoter region of Panc1 cells after crosslinking and pulldown using antibodies against either YAP, TEAD4, or control IgG. Fold enrichment is percent input target gene/percent input Actin. * p < 0.05, ** p < 0.01.

Article Snippet: Global ChIP-seq and UGP2 promoter ChIP-PCR were performed using antibodies against YAP1 (Novus Biologicals NB110-58358) and TEAD4 (ab58310).

Techniques: Staining, Plasmid Preparation, Western Blot, Stable Transfection, Expressing, ChIP-sequencing, Control

Journal: Journal of Biological Chemistry

Article Title: Tumor Suppressor Ras Association Domain Family 5 (RASSF5/NORE1) Mediates Death Receptor Ligand-induced Apoptosis

doi: 10.1074/jbc.m110.165506

Figure Lengend Snippet: FIGURE 2. RASSF5 interacts with MST1 and other components of Hippo signaling pathway. A, U2OS cells cotransfected with FLAG-RASSF5 and eitherMST1,WW45,LATS1,orYAP1wereimmunoprecipitated(IP)witheither FLAG, MST1, WW45, LATS1, or YAP1 antibodies and analyzed by Western blotting (WB). B, 4-week-old mouse brain lysates were immunoprecipitated with anti-RASSF5, anti-MST1, or anti-YAP1 antibody and analyzed by immu- noblotting. C, U2OS cells were transfected with control or MST1, WW45, LATS1, or YAP1 siRNA and treated with varying concentrations of TNF- along with cycloheximide. Cell survival was measured as described.

Article Snippet: Antibodies, Plasmids, and siRNA—The following antibodies were purchased: PARP, Caspase 3, Caspase 8, JNK, phosphoJNK, p38, phospho-p38, p44/42, phospho-p44/42 (Cell SignalingTechnology, Danvers,MA); -actin andTNF-R1 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); anti-FLAG (M2) and BAX (Sigma-Aldrich); and MST1/2, YAP1, and LATS1 (Bethyl Laboratories, Montgomery, TX).

Techniques: Western Blot, Immunoprecipitation, Transfection, Control

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: Genome-wide allelic expression imbalance analysis in osteosarcoma. ( A ) A Manhattan plot showing the result of the genome-wide allelic imbalance analysis. The most significant SNPs were found on pig chromosomes 6, 9, 14 and 16. ( B ) Schematic genomic structure of the YAP1-BIRC3 locus on chromosome 9 in pigs. The blue arrow indicates the position of the 9:33044172 A/G SNP in the 3′UTR of BIRC3 . ( C ) cDNA pyrosequencing result for the SNP 9:33044172 A/G in osteosarcoma (os, n = 48) and matched healthy bone (b) samples collected from flTP53 R167H pigs. To test analysis the validity of the pyrosequencing assay, we used DNA samples ( n = 5) extracted from wild-type pigs. *** p < 0.001.

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: Genome Wide, Expressing, Pyrosequencing Assay

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: YAP1 amplification in p53 deficient osteosarcoma. ( A ) Point plot showing the correlation between 9:33044172 A allele expression and YAP1 copy number. Gray and red points show expression of A allele in bone and OS samples, respectively. Blue points show the measurements in wild-type samples. ( B ) Point plot showing the correlation between 9:33044172 A allele expression and OS ( n = 48) size. ( C ) Point plot showing the correlation between YAP1 copy number and OS ( n = 48) size. ( D ) Quantitative PCR of YAP1 mRNA expression in wild type (wt, n = 5) bones, as well as OS ( n = 48) and matched healthy bone samples from flTP53 R167H pigs. ( E ) Representative Western blot showing YAP1 expression in wild type bone, OS and healthy bone samples from flTP53 R167H pigs. The uncropped Western blots have been shown in . ( F ) Immunohistochemistry staining showing the nuclear location of YAP1 in sections of osteosarcoma from flTP53 R167H pigs. Control samples show staining without the first antibody. Scale bars- 100 μm. (** p < 0.01)

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: Amplification, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining, Control

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: In vitro functional analysis of YAP1 deficiency in p53 deficient primary osteosarcoma cells. ( A ) Sequence analysis showing the result of CRISPR/Cas9 editing of YAP1 in pig OS cells. ( B ) Western blot showing the lack of YAP1 protein in the edited flTP53 R167H OS cells. ( C ) Representative microscopic view showing the morphology of YAP1 −/− /flTP53 R167H OS cells. As a control, flTP53 R167H OS cells were transfected with the GFP control vector (left scale bars, 400μm; right scale bars, 200 μm) ( D ) Proliferation result for YAP1 −/− /flTP53 R167H and flTP53 R167H OS cells. ( E ) Representative microscopic images showing a difference in migration and invasion between YAP1 −/− /flTP53 R167H and flTP53 R167H OS cells (scale bars, 200 μm). Quantitative measurement of migration ( F ) and invasion ( G ). ( H ) Immunofluorescence staining for Ki67 and DAPI in YAP1 −/− /flTP53 R167H and flTP53 R167H OS cells. ( I ) Quantification rates of the Ki67 positive cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: In Vitro, Functional Assay, Sequencing, CRISPR, Western Blot, Control, Transfection, Plasmid Preparation, Migration, Immunofluorescence, Staining

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: Expression of p53 related genes in YAP1 −/− /flTP53 R167H OS cells. ( A ) RT-PCR result for WRAP53, TP53INP1, p14, p16, RB1, TP63, TP73 in YAP1 −/− /flTP53 R167H and flTP53 R167H OS cells. Three independent transfections for each expression vector were performed. NC—negative control. ( B ) Quantitative RT-PCR of p16 mRNA expression. GAPDH mRNA expression was used as a reference. ** p < 0.01. ( C ) Western blot showing lack of p63 expression in YAP1 −/− /flTP53 R167H OS cells.

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: Allelic Expression Imbalance Analysis Identified YAP1 Amplification in p53- Dependent Osteosarcoma

doi: 10.3390/cancers13061364

Figure Lengend Snippet: DNA methylation analysis of the p16 and Rb1 promoter regions in YAP1 −/− /flTP53 R167H OS cells. ( A ) Pyrosequencing result at 8 CpG sites in the p16 promoter region in YAP1 −/− /flTP53 R167H ( n = 3) and flTP53 R167H ( n = 3) OS cells. ( B ) Pyrosequencing result at 9 CpG sites in the Rb1 promoter region in YAP1 −/− /flTP53 R167H ( n = 3) and flTP53 R167H ( n = 3) OS cells. * p < 0.05.

Article Snippet: Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector).

Techniques: DNA Methylation Assay