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

Journal: Clinical Cancer Research

Article Title: Frequency and Genomic Aspects of Intrinsic Resistance to Vismodegib in Locally Advanced Basal Cell Carcinoma

doi: 10.1158/1078-0432.CCR-21-3764

Figure Lengend Snippet: Transcriptional activity and immunohistochemical analysis of the Hedgehog, Hippo-YAP, and WNT markers in BCC. A, Relative activity of the central Hedgehog TF GLI1 based on the expression of its transcription targets in different groups of skin tumors and normal skin. B, RNA expression (logTPM values) of the HHIP and PTCH2 genes which are usually activated in BCC. C, Relative activity of the central HIPPO-YAP TF TEAD1 based on the expression of its transcription targets in different groups of skin tumors and normal skin. D, Quantification of nuclear YAP1 protein expression in tumor cells (% of total tumor cells) among the three groups of samples assessed by YAP1 enzymatic IHC. E, Representative micrographs for YAP1 expression in BCC groups at various magnifications: tumor overview on the whole-slide scan (first column), details of the regions marked with a red square in the first column (20× magnification, second column), and details of regions marked with red square in the second column (40× magnification, third column) with overlapping color mask (fourth column) marking nuclear YAP1-positive cells (red mask). Nuclear YAP1-negative tumor cells are marked in blue. The parts of the image are intentionally duplicated in Supplementary Figs. S2–S4 for S-BCC, IR-BCC, and N-BCC samples, respectively, as the same slides represented. F, Relative activity of the central WNT TF TCF7 based on the expression of its transcription targets in different groups of samples. G, Quantification of membranous BCAT expression in the tumor cells (% of total tumor cells) in the three groups of samples assessed by BCAT enzymatic IHC. H, Representative micrographs for BCAT expression in BCC groups at various magnifications: tumor overview on the whole-slide scan (first column), details of the regions marked with a red square in the first column (20× magnification, second column), and details of regions marked with red square in the second column (40× magnification, third column) with overlapping color mask (fourth column) marking membranous BCAT-positive cells (red mask). BCAT-negative tumor cells are marked in blue and nontumor cells are marked in yellow. The parts of the image are intentionally duplicated in Supplementary Figs. S6–S8 for S-BCC, IR-BCC, and N-BCC samples, respectively, as the same slides represented.

Article Snippet: The next day, slides were incubated for 30 minutes at room temperature with an anti-rabbit biotinylated secondary antibody for YAP1 (catalog number D30-1, GBI Labs; dilution 1:200), and an anti-mouse biotinylated secondary antibody for BCAT (catalog number D30-0, GBI Labs; dilution 1:200), followed by another 30-minute incubation at room temperature with HRP-conjugated streptavidin (catalog number D30-1, GBI Labs; dilution 1:200).

Techniques: Activity Assay, Immunohistochemical staining, Expressing, RNA Expression

Journal: Clinical Cancer Research

Article Title: Frequency and Genomic Aspects of Intrinsic Resistance to Vismodegib in Locally Advanced Basal Cell Carcinoma

doi: 10.1158/1078-0432.CCR-21-3764

Figure Lengend Snippet: Proliferation for ASZ001 Yap1-overexpressing and ASZ001 cell lines for two time intervals (5 and 24 hours). A, ASZ001 cell line in culture (control; left); YAP1-transfected ASZ001 72 hours after transfection (mCherry positivity indicates successful transfection; middle); YAP1-transfected ASZ001 1.5 weeks after transfection and after puromycin treatment for selection of transfected cells (mCherry positivity indicates successful transfection; right). B, Proliferation assay for ASZ001 and ASZ001 YAP1-overexpressing cell lines after 5 and 24 hours of culturing time.

Article Snippet: The next day, slides were incubated for 30 minutes at room temperature with an anti-rabbit biotinylated secondary antibody for YAP1 (catalog number D30-1, GBI Labs; dilution 1:200), and an anti-mouse biotinylated secondary antibody for BCAT (catalog number D30-0, GBI Labs; dilution 1:200), followed by another 30-minute incubation at room temperature with HRP-conjugated streptavidin (catalog number D30-1, GBI Labs; dilution 1:200).

Techniques: Control, Transfection, Selection, Proliferation Assay

Journal: Clinical Cancer Research

Article Title: Frequency and Genomic Aspects of Intrinsic Resistance to Vismodegib in Locally Advanced Basal Cell Carcinoma

doi: 10.1158/1078-0432.CCR-21-3764

Figure Lengend Snippet: Transcriptional activity and immunohistochemical analysis of the Hedgehog, Hippo-YAP, and WNT markers in BCC. A, Relative activity of the central Hedgehog TF GLI1 based on the expression of its transcription targets in different groups of skin tumors and normal skin. B, RNA expression (logTPM values) of the HHIP and PTCH2 genes which are usually activated in BCC. C, Relative activity of the central HIPPO-YAP TF TEAD1 based on the expression of its transcription targets in different groups of skin tumors and normal skin. D, Quantification of nuclear YAP1 protein expression in tumor cells (% of total tumor cells) among the three groups of samples assessed by YAP1 enzymatic IHC. E, Representative micrographs for YAP1 expression in BCC groups at various magnifications: tumor overview on the whole-slide scan (first column), details of the regions marked with a red square in the first column (20× magnification, second column), and details of regions marked with red square in the second column (40× magnification, third column) with overlapping color mask (fourth column) marking nuclear YAP1-positive cells (red mask). Nuclear YAP1-negative tumor cells are marked in blue. The parts of the image are intentionally duplicated in Supplementary Figs. S2–S4 for S-BCC, IR-BCC, and N-BCC samples, respectively, as the same slides represented. F, Relative activity of the central WNT TF TCF7 based on the expression of its transcription targets in different groups of samples. G, Quantification of membranous BCAT expression in the tumor cells (% of total tumor cells) in the three groups of samples assessed by BCAT enzymatic IHC. H, Representative micrographs for BCAT expression in BCC groups at various magnifications: tumor overview on the whole-slide scan (first column), details of the regions marked with a red square in the first column (20× magnification, second column), and details of regions marked with red square in the second column (40× magnification, third column) with overlapping color mask (fourth column) marking membranous BCAT-positive cells (red mask). BCAT-negative tumor cells are marked in blue and nontumor cells are marked in yellow. The parts of the image are intentionally duplicated in Supplementary Figs. S6–S8 for S-BCC, IR-BCC, and N-BCC samples, respectively, as the same slides represented.

Article Snippet: The next day, slides were incubated for 30 minutes at room temperature with an anti-rabbit biotinylated secondary antibody for YAP1 (catalog number D30-1, GBI Labs; dilution 1:200), and an anti-mouse biotinylated secondary antibody for BCAT (catalog number D30-0, GBI Labs; dilution 1:200), followed by another 30-minute incubation at room temperature with HRP-conjugated streptavidin (catalog number D30-1, GBI Labs; dilution 1:200).

Techniques: Activity Assay, Immunohistochemical staining, Expressing, RNA Expression