Journal: Cancer Gene Therapy

Article Title: Atypical cellular responses mediated by intracellular constitutive active TrkB (NTRK2) kinase domains and a solely intracellular NTRK2-fusion oncogene

doi: 10.1038/s41417-024-00809-0

Figure Lengend Snippet: Key resources table.

Article Snippet: Rabbit polyclonal anti- pY397-FAK (1:500) , Cell Signaling , Cat# 3283; RRID: AB_2173659.

Techniques: Western Blot, Recombinant, Staining, Membrane, Bicinchoninic Acid Protein Assay, Gel Extraction, Mutagenesis, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Hippo component YAP promotes focal adhesion and tumour aggressiveness via transcriptionally activating THBS1/FAK signalling in breast cancer

doi: 10.1186/s13046-018-0850-z

Figure Lengend Snippet: YAP was able to induced cell migration, invasion and focal adhesion in breast cancer cell lines . ( a ) Western blot verified the overexpression of YAP in MCF7 cells. EV: empty vector; o/e: overexpression. ( b ) Western blot verified the knockdown of YAP in MDA-MB-231 cells via a collection of siRNAs; siYAP-#2 and siYAP-#3 has relatively high knockdown efficiency, thus these two siRNAs were used in this research. ( c , d ) Transwell assay showing that overexpression of YAP induced cell migration and invasion ability in MCF7 cells. The experiment was performed in triplicate. ** p < 0.01 by Student’s t-test. Scale bar: 100 μm. ( e , f ) Transwell assay showing that knockdown of YAP significantly inhibited cell migration and invasion ability in MDA-MB-231 cells. The experiment was performed in triplicate. ** p < 0.01 by ANOVA test. Scale bar: 100 μm. ( g , h ) Overexpression of YAP induced MCF7 cell adhesion to gelatin. The attached cells were stained with Wright’s-Giemsa and are shown in ( g ). The experiment was performed in triplicate. ** p < 0.01 by Student’s t-test. Scale bar: 100 μm. ( i , j ) Knockdown of YAP significantly inhibited MDA-MB-231 cell adhesion to gelatin. The attached cells were stained with Wright’s-Giemsa and are shown in ( i ). The experiment was performed in triplicate. ** p < 0.01 by Student’s t-test. Scale bar: 100 μm. ( k ) Overexpression of YAP induced focal adhesions in MCF7 cells. Focal adhesions were visualized by co-localization of paxilin (green) and F-actin (stained with phalloidin, red). Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm. ( l ) Knockdown of YAP expression inhibited focal adhesions in MDA-MB-231 cells. Scale bar: 20 μm. ( m ) Quantification of the membrane-localized paxilin in ( k ). The experiment was performed in triplicate. ** p < 0.01 by Student’s t-test. ( n ) Quantification of the membrane-localized paxilin in ( l ). The experiment was performed in triplicate. ** p < 0.01 by ANOVA test

Article Snippet: The membranes were then incubated with the primary antibodies against YAP (Cell Signaling Technology, Cat. #4912), FAK (Abclonal, Cat. #A11131), pY397-FAK (Abclonal, Cat. #AP0302), THBS1 (Abclonal, Cat. #A2125) and GAPDH (Santa Cruz Biotechnology, Cat. #sc-32,233) at a diluted ratio of 1:1000 overnight at 4 °C.

Techniques: Migration, Western Blot, Over Expression, Plasmid Preparation, Knockdown, Transwell Assay, Staining, Expressing, Membrane

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Hippo component YAP promotes focal adhesion and tumour aggressiveness via transcriptionally activating THBS1/FAK signalling in breast cancer

doi: 10.1186/s13046-018-0850-z

Figure Lengend Snippet: YAP-TEAD promoted focal adhesion formation in breast cancer cell lines through inducing FAK phosphorylation. ( a ) Western blot revealed that compared with the YAP-S94A mutant, overexpression of the YAP-S127A mutant could promote FAK Y397 phosphorylation. ( b ) Verteporfin reversed YAP-S127A-induced FAK phosphorylation in MCF7 cells. MCF7-YAP-S127A was treated with verteporfin at a dose of 10 μM for 24 h before the Western blot assay was performed. DMSO was used as a negative control. ( c ) Knockdown of endogenous YAP expression inhibited FAK Y397 phosphorylation in MDA-MB-231 cells. ( d ) Verteporfin inhibited FAK phosphorylation in MDA-MB-231 cells. Cells were treated with verteporfin at a dose of 10 μM for 24 h before Western blot assays were performed. DMSO was used as a negative control. ( e , f ) Western blot verified the inhibition of FAK Y397 phosphorylation via defactinib in MCF7-YAP-S127A (e) and MDA-MB-231 ( f ) cells. Cells were exposed to defactinib at a dose of 10 μM for 8 h before Western blot assays were performed. DMSO was used as a negative control. ( g , h ) Treatment with defactinib (10 μM) for 8 h decreased focal adhesions, both in MCF7-YAP-S127A ( g ) and MDA-MB-231 ( h ). DMSO was used as a negative control. Paxilin (green), F-actin (stained with phalloidin, red). Nuclei (blue). Scale bar: 20 μm. ( i ) Transwell invasion assays showed that exposure to defactinib (10 μM) could significantly reverse YAP-S127A-induced cell invasion in MCF7 cells. DMSO was used as a negative control. The experiment was performed in triplicate. Scale bar: 100 μm. ( j ) Exposure to defactinib (10 μM) significantly decreased cell invasion ability in MDA-MD-231 cells. DMSO was used as a negative control. The experiment was performed in triplicate. Scale bar: 100 μm. ( k ) Quantification of the relative invasion ability in ( i ). ** p < 0.01 by ANOVA test. ( l ) Quantification of the relative invasion ability in ( j ). ** p < 0.01 by Student’s t-test

Article Snippet: The membranes were then incubated with the primary antibodies against YAP (Cell Signaling Technology, Cat. #4912), FAK (Abclonal, Cat. #A11131), pY397-FAK (Abclonal, Cat. #AP0302), THBS1 (Abclonal, Cat. #A2125) and GAPDH (Santa Cruz Biotechnology, Cat. #sc-32,233) at a diluted ratio of 1:1000 overnight at 4 °C.

Techniques: Phospho-proteomics, Western Blot, Mutagenesis, Over Expression, Negative Control, Knockdown, Expressing, Inhibition, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Hippo component YAP promotes focal adhesion and tumour aggressiveness via transcriptionally activating THBS1/FAK signalling in breast cancer

doi: 10.1186/s13046-018-0850-z

Figure Lengend Snippet: YAP-TEAD transcriptionally promoted expression of FAK upstream regulatory factor, thrombospondin 1 (THBS1). ( a ) Analysis of TEAD ChIP-sequence data of MCF7 cells from the ENCODE database (GSM1010860) via the ChIP-seek tool. SL14575 and SL16341 were two bio-replications of the ChIP-sequence data. ( b ) Peaks in promoter-TSS category from ( a ) were exacted and annotated. A total of 192 genes whose promoter was potentially combined with TEAD4 were identified. ( c ) Gene expression profiling was performed in MCF7 cells overexpressing the YAP-S127A mutant compared with empty vector. Expression values of the 192 genes from ( b ) were exacted and presented in a heat map. ( d ) The 192 genes were divided into four categories according to the expression fold change (FC) in MCF7-YAP-S127A vs. MCF7-EV cells. Upregulated: genes that were upregulated by the YAP-S127A mutant with a FC ≥ 2; Unchanged: genes with an expression fold change between the two groups of less than two; Downregulated: genes were downregulated by the YAP-S127A mutant with a FC ≥ 2; Undetected: genes that were not detected by the expression profiling. ( e ) Gene ontology analysis (biological processes) was performed for the 30 upregulated genes from ( d ). “Cell adhesion” was the first enrichment category and contained 6 genes. ( f ) Using the STRING program to analyse potential interactions between FAK (also known as PTK) and the 6 upregulated genes that were included in the “cell adhesion” category (THBS1, HABP2, L1CAM, BCAM, CYR61 and CTGF). THBS1 appeared to be highly correlated to FAK (confidence score: 0.849). ( g ) Dual luciferase reporter assays showed that THBS1 promoter activity could be significantly enhanced by YAP, both in HEK293T and MCF7 cells. * p < 0.05 by Student’s t-test. The experiments were performed in triplicate. ( h ) Dual luciferase reporter assays showed that compared to the YAP-S94A mutant, YAP-S127A could significantly increase THBS1 promoter activity in HEK293T and MCF7 cells. * p < 0.05 and ** p < 0.01 by ANOVA. The experiments were performed in triplicate. ( i ) Through overexpressing the YAP-S127A mutant, the combination of YAP protein and THBS1 promoter was significantly increased in MCF7 cells. Chromatin and proteins were cross-linked, and mouse monoclonal anti-YAP antibodies were used for pulldown. The promoter of THBS1 was amplified and verified via agarose gel electrophoresis. Mouse IgG was used as a negative control. ( j ) Quantitative real-time PCR showed mRNA levels of YAP target genes (CTGF, CYR61) and THBS1 in MCF7-EV, MCF7-YAP-S127A and MCF7-S94A cells. The YAP-S127A mutant could significantly induce THBS1 and YAP target gene expression. GAPDH was used as an internal control. * p < 0.05 by ANOVA. The experiments were performed in triplicate. ( k ) Knockdown of endogenous YAP significantly downregulated THBS1, CTGF and CYR61 expression in MDA-MB-231 cells. GAPDH was used as an internal control. * p < 0.05 by ANOVA. The experiments were performed in triplicate. ( l ) Western blot showed that compared with the YAP-S94A mutant, overexpression of the YAP-S127A mutant significantly induced THBS1 expression. ( m ) Knockdown of endogenous YAP expression significantly decreased THBS1 protein levels in MDA-MB-231 cells. ( n ) Verteporfin could inhibit THBS1 expression in MDA-MB-231 cells. Cells were exposed to verteporfin (10 μM) for 24 h before the Western blot assay was performed. ( o ) THBS1 expression was positively associated with YAP in clinical breast cancer specimens ( R = 0.382, p < 0.01). Gene correlation analysis was based on the TCGA breast invasive carcinoma dataset and was analysed via the R2: Genomics Analysis and Visualization Platform. The degrees of freedom (df) was 1095

Article Snippet: The membranes were then incubated with the primary antibodies against YAP (Cell Signaling Technology, Cat. #4912), FAK (Abclonal, Cat. #A11131), pY397-FAK (Abclonal, Cat. #AP0302), THBS1 (Abclonal, Cat. #A2125) and GAPDH (Santa Cruz Biotechnology, Cat. #sc-32,233) at a diluted ratio of 1:1000 overnight at 4 °C.

Techniques: Expressing, Sequencing, Gene Expression, Mutagenesis, Plasmid Preparation, Luciferase, Activity Assay, Amplification, Agarose Gel Electrophoresis, Negative Control, Real-time Polymerase Chain Reaction, Targeted Gene Expression, Control, Knockdown, Western Blot, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Hippo component YAP promotes focal adhesion and tumour aggressiveness via transcriptionally activating THBS1/FAK signalling in breast cancer

doi: 10.1186/s13046-018-0850-z

Figure Lengend Snippet: YAP triggered FAK phosphorylation and focal adhesion through THBS1. ( a ) Western blot assays revealed that knockdown of THBS1 expression in MCF7-YAP-S127A cells could significantly reverse FAK Y397 phosphorylation. ( b ) Cell adhesion assays showed that knockdown of THBS1 could significantly reverse YAP-S127A-induced cell adhesion in MCF7 cells. The experiments were performed in triplicate. Scale bar: 100 μm. ( c ) Transwell invasion assays showed that knockdown of THBS1 could significantly reverse YAP-S127A-induced cell invasion in MCF7 cells. The experiments were performed in triplicate. Scale bar: 100 μm. ( d ) Quantification of the cell adhesion ability in ( b ). * p < 0.05 and ** p < 0.01 by ANOVA test. ( e ) Quantification of the cell invasion ability in ( c ). ** p < 0.01 by ANOVA test. ( f ) Knockdown of THBS1 inhibited focal adhesion in MCF7-YAP-S127A cells. Red: F-actin (stained with phalloidin); Green: paxilin; Blue: nucleus (stained with DAPI). Scale bar: 20 μm. ( g ) Knockdown of THBS1 reduced FAK Y397 phosphorylation in MDA-MB-231 cells. ( h ) Knockdown of THBS1 expression reduced cell adhesion to gelatin in MDA-MB-231 cells. The experiments were performed in triplicate. Scale bar: 100 μm. ( i ) Transwell invasion assays showed that knockdown of THBS1 expression reduced cell invasion in MDA-MB-231 cells. The experiments were performed in triplicate. Scale bar: 100 μm. ( j ) Quantification of the cell adhesion ability in ( h ). ** p < 0.01 by ANOVA test. ( k ) Quantification of the cell invasion ability in ( i ). ** p < 0.01 by ANOVA test. ( l ) Knockdown of THBS1 reduced focal adhesion in MDA-MB-231 cells. Red: F-actin (stained with phalloidin); Green: paxilin; Blue: nucleus (stained with DAPI). Scale bar: 20 μm. (m) Model for how YAP regulates THBS1 expression and induces focal adhesion

Article Snippet: The membranes were then incubated with the primary antibodies against YAP (Cell Signaling Technology, Cat. #4912), FAK (Abclonal, Cat. #A11131), pY397-FAK (Abclonal, Cat. #AP0302), THBS1 (Abclonal, Cat. #A2125) and GAPDH (Santa Cruz Biotechnology, Cat. #sc-32,233) at a diluted ratio of 1:1000 overnight at 4 °C.

Techniques: Phospho-proteomics, Western Blot, Knockdown, Expressing, Staining