pcdna3 1 myc vector  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: TEAD1 regulates cell proliferation through a pocket-independent transcription repression mechanism

doi: 10.1093/nar/gkac1063

Figure Lengend Snippet: TEAD1 can repress cell proliferation in tumor cells. (A) TEAD1 transcript in 33 tumor lines and the adjacent normal tissues. (B) Visualization and quantification of cell growth in TEAD1 overexpressed cells by TEAD1-P2A (2A self-cleaving peptides)-GFP. (C) Quantification of cell proliferation by EDU incorporation assay. (D) Quantification of apoptosis and cell death by Annexin V and propidium iodide staining. (E) In vivo tumorigenesis in nude mice. The arrows show the tumor xenograft. *P < 0.05, **P < 0.01 and ***P < 0.001; error bars represent standard error of the mean (SEM).

Article Snippet: Myc-tagged human TEAD1 vector was a gift from Kunliang Guan (#33109; Addgene).

Techniques: Staining, In Vivo

Journal: Nucleic Acids Research

Article Title: TEAD1 regulates cell proliferation through a pocket-independent transcription repression mechanism

doi: 10.1093/nar/gkac1063

Figure Lengend Snippet: TEAD1 ablation in pancreatic β cells induces proliferation and an immature phenotype. (A) Random blood glucose levels in β-cell specific TEAD1 knockout (TKO) mice. (B) Glucose (2 mM versus 25 mM) induced insulin secretion in mouse islets isolated from TKO and control mice. (C) TEAD1 immunostaining on pancreas sections from TKO and control mice. (D) Immunoblotting using pancreatic islet lysates isolated from TKO and control mice. (E) Ki67 staining and quantification on pancreas sections from TKO and control mice. (F) Experimental scheme for insulin pump implantation. (G) Ki67 staining and quantification on pancreas sections from insulin pump implanted TKO or control mice. (H) Random blood glucose levels on dpi (days post insulin pump implantation) 7 and 14. (I) Fasting C-peptide levels on dpi 14. (J) Nuclei quantification per islet in TKO and control mice. (K) Relative insulin-positive area (insulin positive area / total area of the pancreas) analysis in TKO and control mice. Total of 32 pancreatic sections from four TKO and four control mice were analyzed. (L) Quantification of cell proliferation by EDU incorporation assay in TEAD1-overexpressed mouse INS2 insulinoma line. *P < 0.05, **P < 0.01 and ***P < 0.001; error bars represent SEM. Con, control.

Article Snippet: Myc-tagged human TEAD1 vector was a gift from Kunliang Guan (#33109; Addgene).

Techniques: Knock-Out, Isolation, Control, Immunostaining, Western Blot, Staining

Journal: Nucleic Acids Research

Article Title: TEAD1 regulates cell proliferation through a pocket-independent transcription repression mechanism

doi: 10.1093/nar/gkac1063

Figure Lengend Snippet: The identification of bona fide TEAD1 target genes. (A) Venn graph showing potential TEAD1 targets (59 genes) by cross-referencing TKO RNA-seq (955 upregulated genes) and TEAD1-ChIP-seq (392 genes) datasets. (B) Narrowing down to nine TEAD1 direct target genes (TTG) based on regulatory sequence proximity to transcription start sites (TSS) (<1000 bp). (C) TEAD1-ChIP-seq in pancreatic progenitor cells showing strong TEAD1-bound signals that are close to TSS of the candidate TTGs. (D) TEAD1-ChIP-seq in wild-type mouse islets showing TEAD1-bound signals that are close to TSS on WWC2, NR4A3, Amotl2, and LATS2 genes. (E) Quantitative PCR showing the expression of TTGs in TKO and control islets isolated from 12-week-old male mice. (F) Illustration of human CTGF promoter (hCTGF) driven luciferase reporter and mutant human CTGF promoter (ΔhCTGF) driven luciferase reporter on which the MCAT motif was moved to the reverse strand. A luciferase assay showing no difference in activity between ΔhCTGF and hCTGF promoters with co-transfection of YAP5SA and TEAD1. (G) Luciferase assays using mouse YAP1 promoter reporter (mYAP1r) show that TEAD1 and TEAD1 + VGLL4 repress YAP1 transcription, while YAP5SA promotes YAP1 transcription. (H) Split-GFP system showing no binding as indicated by GFP signal between ΔTEAD1 (truncated TEAD1) and YAP1/TAZ/VGLL4. mCherry signal indicate transfection efficiency. Nuclei were counterstained with diamidino-2-phenylindole (DAPI) (blue). (I) mYAP1r-promoter luciferase assays show both TEAD1 and ΔTEAD1 repress YAP1 transcription. (J) ΔmYAP1r (MCATs mutant)-promoter luciferase assays show absent repression of YAP1 transcription by TEAD1 or ΔTEAD1 whereas YAP5SA transcriptional activation of YAP1 is impaired. (K) Human YAP1 promoter and (L) Human TEAD3 promoter luciferase assays show TEAD1 and ΔTEAD1 repression and YAP5SA activation of transcription. (M) ΔTEAD1 inhibit HeLa cell growth. GFP positivity demonstrate ΔTEAD1 or backbone (empty vector) lentiviral transduction. (N) YAP1 protein expression by Western blotting after TEAD1 and ΔTEAD1 overexpression in Hela cells. (O) Human TTGs promoter luciferase assay show TEAD1 and ΔTEAD1 repress the transcription of most TTGs except KNTC1, while YAP5SA promotes the transcription of all TTGs. *P < 0.05, **P < 0.01 and ***P < 0.001; error bars represent SEM.

Article Snippet: Myc-tagged human TEAD1 vector was a gift from Kunliang Guan (#33109; Addgene).

Techniques: RNA Sequencing, ChIP-sequencing, Sequencing, Real-time Polymerase Chain Reaction, Expressing, Control, Isolation, Luciferase, Mutagenesis, Activity Assay, Cotransfection, Binding Assay, Transfection, Activation Assay, Plasmid Preparation, Transduction, Western Blot, Over Expression

Journal: Nucleic Acids Research

Article Title: TEAD1 regulates cell proliferation through a pocket-independent transcription repression mechanism

doi: 10.1093/nar/gkac1063

Figure Lengend Snippet: Function validation of TTGs. (A) HOPFLASH reporter luciferase assay show WWC2, AMOTL2, WTIP and YAP5SA regulation of Hippo signaling activity. (B) Human Ki67-promoter reporter (hKi67r) construct. (C) hKi67r reporter activities suggest differential regulatory effects of proliferation by TTGs. (D) Flow cytometry analysis of proliferative (EDU+) HeLa cells after TTGs overexpression (GFP+); double positives are indicated by grey dots. (E) Quantification of flow cytometry analysis in TTGs overexpressed INS2 cells. (F) Schematic representation of NR4A3 isoforms: NR4A3L, full-length NR4A3; NR4A3S, short form of NR4A3. (G) Quantitative PCR showing NR4A3L and NR4A3S mRNA expression in TKO islets. (H) Quantitative PCR showing Ins1, Ins2, Ki67, NR4A3L and NR4A3S mRNA expression in INS2 cells after TEAD1 overexpression (TEAD1OV). (I) Immunostaining and quantification of Ki67 + Insulin + cells in NR4A3L- and NR4A3S-overexpressing mouse islets. (J) Quantitative PCR showing Ki67, Ins1, Ins2, PDX1, MAFA and UCN3 mRNA expression in NR4A3L- and NR4A3S-overexpressed INS2 cells. (K) hKi67r reporter activity show WTIP, WWC2, AMTOL2, PKCiota and NR4A3 antagonize the proliferation effect of YAP5SA while co-transfecting with YAP5SA at a 1:1 molar ratio in HeLa cells. *P < 0.05, **P < 0.01 and ***P < 0.001; error bars represent SEM.

Article Snippet: Myc-tagged human TEAD1 vector was a gift from Kunliang Guan (#33109; Addgene).

Techniques: Biomarker Discovery, Luciferase, Activity Assay, Construct, Flow Cytometry, Over Expression, Real-time Polymerase Chain Reaction, Expressing, Immunostaining

Journal: Nucleic Acids Research

Article Title: TEAD1 regulates cell proliferation through a pocket-independent transcription repression mechanism

doi: 10.1093/nar/gkac1063

Figure Lengend Snippet: TEAD1 knockout in mouse pancreatic β cells results in elevated YAP1 expression. (A) Western blotting demonstrating YAP1 expression in TKO mouse islets. (B) Detection of YAP1 expression on TKO and control mouse pancreas sections (the arrows indicate YAP1 nuclear staining). (C) Immunostaining on isolated TKO islets showing ex vivo verteporfin effects on proliferation, with quantification showing in the bar chart. (D) Luciferase promoter assay showing TEAD pathway activities in TKO islets. (E) Volcano plot highlighting CCN2 (CTGF) transcript expression in TKO islets. (F) Volcano plot highlighting YAP1 transcript expression in the myocardium of TEAD1 cardiomyocyte-specific knockout mice. *P < 0.05, **P < 0.01 and ***P < 0.001; error bars represent SEM.

Article Snippet: Myc-tagged human TEAD1 vector was a gift from Kunliang Guan (#33109; Addgene).

Techniques: Knock-Out, Expressing, Western Blot, Control, Staining, Immunostaining, Isolation, Ex Vivo, Luciferase, Promoter Assay

Journal: Nucleic Acids Research

Article Title: TEAD1 regulates cell proliferation through a pocket-independent transcription repression mechanism

doi: 10.1093/nar/gkac1063

Figure Lengend Snippet: TEAD1 prevents RNA-polymerase II (POLII) from binding to DNA. (A) Two potential POLII binding sites (checkpoints, CK) in YAP1 reporter vector were chosen to perform POLII-ChIP experiments. (B) ChIP assay showing lower POLII binding on the two CKs after TEAD1 overexpression. (C) ChIP assay showing lower POLII binding on the endogenous YAP1 and NR4A3 promoter region after TEAD1 overexpression in HeLa cells. (D) Western blotting showing POLII expression in TEAD1-overexpressing or empty vector transduced HeLa cells. (E) Schematic representation showing the TEAD1-TTG regulatory loop in pancreatic β cells. *P < 0.05, **P < 0.01 and ***P < 0.001; error bars represent SEM.

Article Snippet: Myc-tagged human TEAD1 vector was a gift from Kunliang Guan (#33109; Addgene).

Techniques: Binding Assay, Plasmid Preparation, Over Expression, Western Blot, Expressing

Journal: Cell reports

Article Title: ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function

doi: 10.1016/j.celrep.2022.110598

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To generate Myc- and Halo-tagged KIF5A constructs, a C-terminal Halo tag was first cloned by PCR and ligated into HindIII/AflII sites in the pRK5:Myc-KIF5A vector (Addgene clone #127616).

Techniques: Recombinant, Transfection, Purification, Modification, Lysis, Blocking Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Expressing, Clone Assay, Plasmid Preparation, Sequencing, Immunoprecipitation, RNA Sequencing Assay, Mass Spectrometry, Software, Imaging, Microscopy, Magnetic Beads, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function

doi: 10.1016/j.celrep.2022.110598

Figure Lengend Snippet: (A) KIF5A domain structure. The kinesin light chain domain, the hinge domain, and the regulatory IAK domain are indicated. Arrows in the expanded intron/exon diagram indicate the ALS-related mutations. A mutation denoted with −14, is positioned 14 bp upstream of exon 27, but still creates the same mutant C terminus. Image created with Biorender.com.

Article Snippet: To generate Myc- and Halo-tagged KIF5A constructs, a C-terminal Halo tag was first cloned by PCR and ligated into HindIII/AflII sites in the pRK5:Myc-KIF5A vector (Addgene clone #127616).

Techniques: Mutagenesis

Journal: Cell reports

Article Title: ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function

doi: 10.1016/j.celrep.2022.110598

Figure Lengend Snippet: (A) SKNAS cells expressing V5-tagged KIF5AΔExon27 show increased microtubule (MT) co-localization compared with KIF5AWT as demonstrated by V5-KIF5A highlighting the MT tracks. Examples of KIF5A (V5; green) and β-tubulin (red) co-localization are indicated by arrowheads. Many cells have KIF5AΔExon27-associated MTs with a non-radial pattern (asterisks). Scale bars, 10 mm (wide view), 5 mm (enlargement).

Article Snippet: To generate Myc- and Halo-tagged KIF5A constructs, a C-terminal Halo tag was first cloned by PCR and ligated into HindIII/AflII sites in the pRK5:Myc-KIF5A vector (Addgene clone #127616).

Techniques: Expressing

Journal: Cell reports

Article Title: ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function

doi: 10.1016/j.celrep.2022.110598

Figure Lengend Snippet: (A) Schematic representation of the single-molecule labeling method used to track KIF5A axonal movement.

Article Snippet: To generate Myc- and Halo-tagged KIF5A constructs, a C-terminal Halo tag was first cloned by PCR and ligated into HindIII/AflII sites in the pRK5:Myc-KIF5A vector (Addgene clone #127616).

Techniques: Labeling

Journal: Cell reports

Article Title: ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function

doi: 10.1016/j.celrep.2022.110598

Figure Lengend Snippet: (A) Mass spectrometry analysis of V5-tagged KIF5AWT and KIF5AΔExon27 bound proteins in SKNAS cells. Venn diagram indicates the number of protein binding partners altered in KIF5AΔExon27 mutant immunoprecipitations. Yellow region: proteins that are unique to, or have ≥4× increase in the amount bound to, KIF5AΔExon27. Red region: proteins that are absent from, or have ≥4× decrease in the amount bound to, KIF5AΔExon27. Orange region: proteins that show no binding preference to either form of KIF5A.

Article Snippet: To generate Myc- and Halo-tagged KIF5A constructs, a C-terminal Halo tag was first cloned by PCR and ligated into HindIII/AflII sites in the pRK5:Myc-KIF5A vector (Addgene clone #127616).

Techniques: Mass Spectrometry, Protein Binding, Mutagenesis, Binding Assay

Journal: Cell reports

Article Title: ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function

doi: 10.1016/j.celrep.2022.110598

Figure Lengend Snippet: (A) Patient-derived Arg1007Lys mutant KIF5A iPSC line and isogenic control differentiated into motor neurons (iMNs) display the MN specific marker, Islet1/2 (red), and Tuj1 (white) at DIV15. Scale bar, 50 μm.

Article Snippet: To generate Myc- and Halo-tagged KIF5A constructs, a C-terminal Halo tag was first cloned by PCR and ligated into HindIII/AflII sites in the pRK5:Myc-KIF5A vector (Addgene clone #127616).

Techniques: Derivative Assay, Mutagenesis, Marker

Journal: Cell reports

Article Title: ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function

doi: 10.1016/j.celrep.2022.110598

Figure Lengend Snippet: ALS-related KIF5A mutations lead to defective autoinhibition (I). As a result, KIF5A has increased binding to MTs and altered axonal transport (II), MT remodeling (III), and growth cone accumulation (IV). The protein and RNA binding partners of mutant KIF5A are also changed (V). On a global scale, differences in gene expression (VI) occur as well as NCT disruptions (VIII) which may affect gene splicing (VII). Ultimately the disruption of cellular homeostasis leads to cellular toxicity and death. This image was created with BioRender.com.

Article Snippet: To generate Myc- and Halo-tagged KIF5A constructs, a C-terminal Halo tag was first cloned by PCR and ligated into HindIII/AflII sites in the pRK5:Myc-KIF5A vector (Addgene clone #127616).

Techniques: Binding Assay, RNA Binding Assay, Mutagenesis, Expressing

Journal: Cell reports

Article Title: ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function

doi: 10.1016/j.celrep.2022.110598

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To generate Myc- and Halo-tagged KIF5A constructs, a C-terminal Halo tag was first cloned by PCR and ligated into HindIII/AflII sites in the pRK5:Myc-KIF5A vector (Addgene clone #127616).

Techniques: Recombinant, Transfection, Purification, Modification, Lysis, Blocking Assay, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Expressing, Clone Assay, Plasmid Preparation, Sequencing, Immunoprecipitation, RNA Sequencing Assay, Mass Spectrometry, Software, Imaging, Microscopy, Magnetic Beads, Real-time Polymerase Chain Reaction