Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: YAP1 plays a key role of the conversion of normal fibroblasts into cancer-associated fibroblasts that contribute to prostate cancer progression

doi: 10.1186/s13046-020-1542-z

Figure Lengend Snippet: YAP1 activates actin and cytoskeletal proteins to transform NFs to CAFs by regulating SRC. a The association of YAP1 and SRC in prostate cancer was analysed online at http://gepia.cancer-pku.cn/ . Pearson R = 0.32. b - c The mRNA expression levels of YAP1, α-SMA and SRC in the indicated four cell lines were detected by qRT-PCR. d The protein expression levels of YAP1, α-SMA, SRC and p-SRC in the indicated four cell lines were detected by western blot. GAPDH was used as an endogenous reference gene. e Western blot was used to detect the protein expression levels of TEAD1, YAP1, p-YAP1, SRC, p-SRC and α-SMA after siTEAD1 transfection of the CAFs. GAPDH was used as an endogenous reference gene. f Western blot was used to detect the protein expression levels of TEAD1, YAP1, p-YAP1, SRC, p-SRC and α-SMA when they were knocked down or overexpressed in CAFs. GAPDH was used as an endogenous reference gene. g The interaction between YAP1 and TEAD1 in the CAFs was determined by the co-IP assay. The relative levels of YAP1 or TEAD1 in these cells were determined by western blot using a YAP1 or TEAD1 antibody. h Chromatin immunoprecipitation (ChIP) of the CAFs was performed with control IgG and TEAD1 antibodies. The precipitation of the SRC promoter was examined by PCR. i A dual-luciferase reporter assay driven by the SRC promoter was co-transfected in the presence or absence of YAP1 or TEAD1. The relative luciferase activities were determined by calculating the ratio of firefly luciferase activities over Renilla luciferase activities. Three independent experiments were conducted, with the means±s.d. of the relative luciferase activities shown. j Western blot was used to detect the protein expression levels of SRC, YAP1, TEAD1, MYL9, F-actin, paxillin and α-SMA after siSRC transfection of the CAFs. GAPDH was used as an endogenous reference gene. k qRT-PCR detection of mRNA expression levels of MYL9, F-actin and paxillin in the CAFshYAP1 group. l Western blot was used to detect the protein expression levels of SRC, MYL9, F-actin and paxillin in the CAFshYAP1 group. GAPDH was used as an endogenous reference gene

Article Snippet: The following shRNA plasmids were used in this study for in vitro transfection: YAP1 Mouse shRNA Plasmid, CAT#: TG502437, Origene; TEAD1 Mouse shRNA Plasmid, CAT#: TL513813, Origene; shRNA vector, CAT#: TR30007, Origene; YAP1 Mouse Tagged ORF Clone, CAT#: MR226049, TrueORF®; and TEAD1 Mouse Tagged ORF Clone, CAT#: MR206462, TrueORF®.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation, Luciferase, Reporter Assay

Journal: Nature Communications

Article Title: Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma

doi: 10.1038/s41467-018-06258-2

Figure Lengend Snippet: TEAD is the top selectively enriched motif at GSC-specific open chromatin and TEAD1 is its most highly expressed family member across GBMs a , b Homer de novo motif discovery outlines the 20 most highly enriched TF motifs at chromatin accessibility regions defined by the GSC tumor-specific ( a ) and developmentally shared ( b ) differential ATAC-seq peak analyses (motifs in bold show selective enrichment in only one peak set). The TEAD motif (with highest scores for TEAD4 and TEAD1) is the top, selectively enriched motif within differential GSC tumor-specific peaks (in red). See also Supplementary Data . c Bar graph of rld-normalized gene expression values for all significantly and uniquely enriched GSC tumor-specific TF motifs, generated from parallel RNA-seq data in E+GSC and E−GBM populations. TEAD1 is the only highly expressed gene (top 25th percentile), which is differentially overexpressed in E+GSCs (* p = 0.0496 for TEAD1, ** p = 0.006 for ZNF416, n = 3. Bars represent mean ± SEM). d Violin plot of TEAD1-4 expression in TCGA GBM RNA-seqV2 data ( n = 150) shows that TEAD1 is the most highly expressed TEAD family member, followed by TEAD2 , TEAD3 , and TEAD4 . Expression shown is log2(normalized counts + 1), normalized as detailed in methods. p -Values from one-sided Wilcoxon matched pairs test. Bar represents median value. e Bar graph of rld-normalized gene expression values for TEAD1-4 derived from RNA-seq E + GSC data (*** p < 0.001; n = 3. Bars represent mean ± SEM)

Article Snippet: For overexpression studies, the (Myc-DDK-tagged)-TEAD1, -AQP4, and CDH11 human ORF lentivirus constructs were transfected and purified from Lenti-X 293T cells (Origene RC215492L1, RC204693L1, and RC203810L1, respectively).

Techniques: Expressing, Generated, RNA Sequencing Assay, Derivative Assay

Journal: Nature Communications

Article Title: Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma

doi: 10.1038/s41467-018-06258-2

Figure Lengend Snippet: CRISPR-Cas9 ablation of TEAD1/4 inhibits migration in primary GBM cells. a Western immunoblot confirms population knockout of TEAD1 and TEAD4 after CRISPR-Cas9-mediated gene ablation. b Cell growth analysis reveals significantly decreased proliferation in TEAD1KO cells at 48–72 h, compared to sham ( n = 3; 48 h: ** p = 0.008; 72 h: * p = 0.01. Bars represent mean ± SEM). c Neurosphere (NS) assays show no difference in sphere number (day 6; n = 3 wells, multiple NS per well. Bars represent mean ± SEM). d Neurosphere (NS) assays show decreased sphere size in TEAD1 knockout, compared to sham. (day6; n = 3 wells, multiple NS per well; TEAD1KO: ** p = 0.002. Dots represent individual NS and lines delineate mean). e Transwell invasion assays show decreased percent cell invasion in TEAD1KO and TEAD4KO cells, compared to sham (24 h; n = 3 wells; TEAD1KO: ** p = 0.002; TEAD4KO: ** p = 0.001. Bars represent mean ± SEM). On right, representative images of transwell invasion chamber membranes are shown. f , g Spheroid migration assays show decreased area of confluent cell migration (dispersion) at 36 h in TEAD1KO and TEAD4KO cells, compared to sham ( f , PDL substrate), with partial rescue of migratory deficits in TEAD1KO cells after TEAD1 overexpression (OE) ( g , laminin + PDL substrate) ( n = 3 wells, with multiple NS per well; TEAD1KO: *** p = 0.0001; TEAD4KO: *** p = 0.00007; TEAD1OE 1: *** p = 0.00058; TEAD1OE 2 (1/10 dilution of TEAD1OE 1): *** p = 0.00015. Bars represent mean ± SEM). On right, migration area marked by red dash line in representative spheroids is shown. All experiments in a – g are performed on G-13063 cells. Scale bars = 75 μM. See also Supplementary Fig.

Article Snippet: For overexpression studies, the (Myc-DDK-tagged)-TEAD1, -AQP4, and CDH11 human ORF lentivirus constructs were transfected and purified from Lenti-X 293T cells (Origene RC215492L1, RC204693L1, and RC203810L1, respectively).

Techniques: CRISPR, Migration, Western Blot, Knock-Out, Over Expression

Journal: Nature Communications

Article Title: Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma

doi: 10.1038/s41467-018-06258-2

Figure Lengend Snippet: In vivo infiltration is highly impaired in TEAD1KO GBM xenografts. a Representative immunofluorescent histology of tumor engraftment and core growth near the injection site in sham and TEAD1KO cells, 3.5 months after orthotopic xenotransplantation. b Representative immunofluorescence images of TEAD1 expression, confirming the stable loss of TEAD1 in TEAD1KO xenografts. Insets represent the entire TEAD1 immunofluorescence image with DAPI co-labeling. c Quantification of cell proliferation in sham and TEAD1KO cells at 3.5 months post xenotransplantation ( n = 3 animals per condition; Ki67+ cells counted out of all HNA+ cells in 11 serial histological sections. Bars represent mean ± SEM). d Quantification of migratory tumor spread in sham and TEAD1KO cells at 3.5 months post xenotransplantation ( n = 3 animals per condition; *** p = 0.0003 for both. Area = sum of areas with tumor spread in 11 serial coronal sections. Volume = average area × the distance between the first and the last serial section examined. Bars represent mean ± SEM). Schematic of the analyzed mouse brain serial coronal sections is depicted on the right. e Representative example of infiltrative tumor spread along the corpus callosum in sham and TEAD1KO cells at 3.5 months post xenotransplantation. HNA: human nuclear antigen, CC: corpus callosum, v: ventricle, a anterior, p: posterior. Scale bar = 50 μM

Article Snippet: For overexpression studies, the (Myc-DDK-tagged)-TEAD1, -AQP4, and CDH11 human ORF lentivirus constructs were transfected and purified from Lenti-X 293T cells (Origene RC215492L1, RC204693L1, and RC203810L1, respectively).

Techniques: In Vivo, Injection, Immunofluorescence, Expressing, Labeling

Journal: Nature Communications

Article Title: Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma

doi: 10.1038/s41467-018-06258-2

Figure Lengend Snippet: Validation of TEAD1-binding targets by chromatin immunoprecipitation. a Density plot of correspondence analysis between chromatin accessibility and gene expression. Plotted on the y -axis is the average rld-normalized gene expression for each gene from all RNA-seq E+GSC sample data. Plotted on the x -axis is the highest ATAC-seq peak associated with the proximal promoter [−5 kb, +3 kb] of the same gene. Color intensity indicates density of the gene population, with red representing higher densities and blue representing lower densities. Strong correspondence is observed between open chromatin peaks and a moderate/high level of gene expression in all E+GSC samples (plot for one representative sample shown here). Several putative TEAD1-target genes of interest are indicated on the plot. b IGV plot of ATAC-seq piled reads at EGFR , AQP4 , and CDH4 in four different acutely sorted E+GSCs (D.PROM distal promoter of EGFR, peak180759: chr7:55,000,372–55,001,595; P.PROM proximal promoter to TSS of EGFR, peak180777: chr7:55,085,981–55,088,747). For this IGV representation, reads are centered on the cut site of the Tn5 enzyme, correcting for the 9 bp occupancy of Tn5, and presumed footprints/peak troughs corresponding to TEAD motifs are delineated by downward arrow. c Chromatin immunoprecipitation (ChIP-PCR) in GBM tissues. Significant enrichment of TEAD1 (but not TEAD4) over IgG is seen specifically at differential open chromatin peaks with associated TEAD1 motif within the upstream promoter of EGFR (D. PROM, chr7:55001141 motif start site) ( n = 6, * p = 0.011), at CDH4 (chr20:60011935 motif start site) ( n = 6, * p = 0.027), and at the proximal promoter of AQP4 (chr18:24444251 motif start site) ( n = 6, * p = 0.029). Significant binding above background is not observed at EGFR P.PROM or at chromatin inaccessible regions (IN2 = EGFR intron 2). Enrichment is expressed as fold increase over IgG, after normalization with 10% input, using 2^–ΔΔCt analysis accounting for primer efficiency: ( E ^IA− S ) sample /( E ^IA− S ) IgG . E : primer efficiency; IA: 10% Input-Adjusted Ct; S : sample Ct. Bars represent mean ± SEM. d Western immunoblot illustrates decreased expression of AQP4 and CDH4 in TEAD1KO but not in TEAD4KO G-13063 GBM cells

Article Snippet: For overexpression studies, the (Myc-DDK-tagged)-TEAD1, -AQP4, and CDH11 human ORF lentivirus constructs were transfected and purified from Lenti-X 293T cells (Origene RC215492L1, RC204693L1, and RC203810L1, respectively).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Expressing, RNA Sequencing Assay, Western Blot

Journal: Nature Communications

Article Title: Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma

doi: 10.1038/s41467-018-06258-2

Figure Lengend Snippet: TEAD1 regulates expression of EGFR. a Immunofluorescence image depicts observed expression of EGFR in sham and TEAD1-knockout G-13063 xenografts 3.5 months post transplantation. Scale bar = 50 μM. b Western immunoblot depicts marked downregulation of EGFR in vitro after knockout of TEAD1, but not TEAD4, in G-13063 cells, which is partially restored after TEAD1 overexpression for 48 h. On right is a bar graph quantification of immunoblots from three independent experiments ( n = 3; TEAD1KO vs. TEAD4KO: ** p = 0.004 and ** p = 0.003 for TEAD1 and EGFR, respectively; TEAD1KO + TEAD1OE vs. TEAD1KO: * p = 0.01 for TEAD1; p = 0.068 for EGFR in one tail t -test analysis. Bars represent mean ± SEM). c Western immunoblot depicts downregulation of pERK/ERK but not pAKT/AKT in TEAD1KO cells, compared to sham ( n = 3 cell lines; pERK/ERK: * p = 0.029; bars represent mean ± SEM). On right are shown representative immunoblot images from one cell line

Article Snippet: For overexpression studies, the (Myc-DDK-tagged)-TEAD1, -AQP4, and CDH11 human ORF lentivirus constructs were transfected and purified from Lenti-X 293T cells (Origene RC215492L1, RC204693L1, and RC203810L1, respectively).

Techniques: Expressing, Immunofluorescence, Knock-Out, Transplantation Assay, Western Blot, In Vitro, Over Expression

Journal: Nature Communications

Article Title: Analysis of chromatin accessibility uncovers TEAD1 as a regulator of migration in human glioblastoma

doi: 10.1038/s41467-018-06258-2

Figure Lengend Snippet: TEAD1 regulates GBM migration by modulating AQP4 expression. a Venn diagram depicts the intersection of genes with TEAD motifs and genes consistently downregulated in TEAD1KO cell lines and migration-deficient spheroids (striped area). AQP4 is the only one of 32 genes in this intersection found to be a direct TEAD1 binding target in vivo (highlighted in red). ATAC-seq set contains 2612 peak-annotated genes from E+GSC vs. E−GBM+NSPC differential accessibility analysis. Top RNA-seq set (overall targets) contains all 1648 significantly up or downregulated genes from TEAD1KO vs. Sham differential expression analysis in all samples from four different patient-derived GBM lines and three migration experiments ( n = 7; p adj. < 0.05; all log2(fold change) values of HGNC-annotated genes included). Bottom RNA-seq set (migratory targets) contains 865 significantly up or downregulated genes from TEAD1KO vs. Sham G-13063 spheroids from three independent migration experiments ( n = 3; p adj. < 0.05; log2(fold change) >1 or <−1). See also Supplementary Fig. . b Spheroid migration assay showing significant reversal of cell dispersion deficit at 30 h in TEAD1 knockout cells after overexpression of CDH11 or AQP4 (G-13063 line; laminin + PDL substrate; n = 3 wells. TEAD1KO + CDH11OE: ** p = 0.004; TEAD1KO + AQP4OE: ** p = 0.0015. Bars represent mean ± SEM). On right, migration area marked by red dash line in representative spheroids is shown. Scale bar = 75 μM. c Quantification of AQP4 expression by RT-qPCR. AQP4 is significantly upregulated in TEAD1KO cells after TEAD1 overexpression ( n = 6, 4 technical and 2 biological replicates; * p = 0.02 TEAD1KO + TEAD1OE vs. TEAD1KO) and is robustly expressed after exogenous lentivirus overexpression. Bars represent mean ± SEM

Article Snippet: For overexpression studies, the (Myc-DDK-tagged)-TEAD1, -AQP4, and CDH11 human ORF lentivirus constructs were transfected and purified from Lenti-X 293T cells (Origene RC215492L1, RC204693L1, and RC203810L1, respectively).

Techniques: Migration, Expressing, Binding Assay, In Vivo, RNA Sequencing Assay, Derivative Assay, Knock-Out, Over Expression, Quantitative RT-PCR