pgama yap plasmid  (Addgene inc)

Journal: bioRxiv

Article Title: YAP1 Contributes to The Development of Contractile Force and Sarcomere Maturation in Human Pluripotent Stem Cell-Derived Cardiomyocytes

doi: 10.1101/2024.07.02.601803

Figure Lengend Snippet: Principal component analysis (PCA) (left) and heatmap representation (right) of the differentially expressed genes (DEG) in WT and YAP1-KO hESC-CMs as identified by RNA-sequencing (day 15 of differentiation, n=3) (A). Lollipop plot representation of differential gene expression of key CM maturation genes in YAP1-KO compared to WT hESC-CMs (n=3) (B). Bar plot representation of percentage of EdU-positive (left) and troponin-T positive (right) YAP1-KO and WT hESC- CMs (n=3) (C). Venn diagram representation of YAP1 bona fide targets, as the genes both identified as YAP1 targets in ChIP-seq analysis and differentially expressed in YAP1-KO RNA-seq (D). Bar plot representation of percentage of bona fide genes with YAP1 peaks in different genomic regulatory sequences (E). Bar plot representation of frequency of YAP1 binding to indicated regulatory genomic sequences (F). Lollipop plot representation of gene expression of the top 10 differentially regulated bona fide genes grouped by genomic regulatory sequences (G).

Article Snippet: Plasmid containing full length YAP1 with T2A-mCherry tag (#74942, Addgene) was transfected together with envelope expressing plasmid 9 - PMS2.G (#12259, Addgene), and empty backbone packaging plasmid PSPAX2 (#12260, Addgene) into HEK293T cells using FuGENE® HD Transfection Reagent according to manufacturer’s instructions.

Techniques: RNA Sequencing, Gene Expression, ChIP-sequencing, Binding Assay, Genomic Sequencing

Journal: bioRxiv

Article Title: YAP1 Contributes to The Development of Contractile Force and Sarcomere Maturation in Human Pluripotent Stem Cell-Derived Cardiomyocytes

doi: 10.1101/2024.07.02.601803

Figure Lengend Snippet: Lollipop plot representation of up to 10 most differentially expressed (|Log2FC|) genes of indicated gene ontology (GO) categories in YAP1-KO compared to WT hESC-CMs (n=3) (A). Western blot analysis of the indicated sarcomeric proteins in YAP1-KO and WT CMs quantification on bottom; GAPDH was used for normalization (B). Characterization of changes in morphology and sarcomere length in WT and YAP1-KO CMs at days 10, 15, and 30 of differentiation. Representative segmentation of α-sarcomeric actinin into organized (magenta) and disorganized (blue) structures (C). Morphometry of projected cell area, length, width (D), and sarcomere length (E). Statistics: Two way ANOVA, Interaction: P <0.0001. Violin plot representation of projected cell area (left) and sarcomere length (right) of YAP1-KO CMs and YAP1-KO CMs rescued by ectopically expressed YAP1 T2A-mCherry plasmid (Qin et al. 2016) (F). hESC-WT CMs on day 16 were untreated (Control) or incubated with latrunculin A. Analysis of YAP1 localization by nuclear/cytoplasmic (n/c) ratio of YAP1 intensity and sarcomere length (G). The data are shown as mean±S.D. (N=3). Statistics: Unpaired t test, ns: P >0.05, * P <0.05, **** P <0.0001.

Article Snippet: Plasmid containing full length YAP1 with T2A-mCherry tag (#74942, Addgene) was transfected together with envelope expressing plasmid 9 - PMS2.G (#12259, Addgene), and empty backbone packaging plasmid PSPAX2 (#12260, Addgene) into HEK293T cells using FuGENE® HD Transfection Reagent according to manufacturer’s instructions.

Techniques: Western Blot, Plasmid Preparation, Control, Incubation

Journal: bioRxiv

Article Title: YAP1 Contributes to The Development of Contractile Force and Sarcomere Maturation in Human Pluripotent Stem Cell-Derived Cardiomyocytes

doi: 10.1101/2024.07.02.601803

Figure Lengend Snippet: Lollipop plot representation of differential gene expression of indicated gene categories in YAP1-KO compared to WT hESC-CMs (n=3) (A). Representative action potentials (B, left) and quantification of AP parameters in WT (n=12) and YAP1-KO (n=8) CMs: beating rate, maximum diastolic potential (MDP), corrected AP duration at 50% of repolarization (cAPD 50 ) (B, right). Representative recordings of pacemaker current (I f ) (C, left), I f I/V relations and steady-state activation curves (left) in WT (n=23) and YAP1-KO (n=14) CMs (C, right). Representative recordings (D, left) and quantification of peak T-type Ca 2+ current (I CaT ) at −20 mV in WT (n=16) and YAP1-KO (n=16) CMs (D, right). Representative recordings of long lasting Ca 2+ current (I CaL ) (E, left), ICaL I/V relations and steady-state activation/inactivation curves in WT (n=23) and YAP1-KO (n=18) CMs (E, right). Representative recordings of sodium current (INa) (F, left), I Na I/V relations and steady-state activation/inactivation curves in WT (n=12) and YAP1-KO (n=18) CMs (F, right). Statistics: unpaired t test, * P <0.05.

Article Snippet: Plasmid containing full length YAP1 with T2A-mCherry tag (#74942, Addgene) was transfected together with envelope expressing plasmid 9 - PMS2.G (#12259, Addgene), and empty backbone packaging plasmid PSPAX2 (#12260, Addgene) into HEK293T cells using FuGENE® HD Transfection Reagent according to manufacturer’s instructions.

Techniques: Gene Expression, Activation Assay

Journal: bioRxiv

Article Title: YAP1 Contributes to The Development of Contractile Force and Sarcomere Maturation in Human Pluripotent Stem Cell-Derived Cardiomyocytes

doi: 10.1101/2024.07.02.601803

Figure Lengend Snippet: Representative traces of voltage-induced Ca 2+ transient (CaT) (A, left) and quantification of CaT parameters: time to peak (TTP), amplitude, and decay kinetics at 20%, 50%, 90% decay time in WT (n=29) and YAP1-KO (n=23) CMs (A, right). Representative traces of caffeine-induced CaT (B, left) and quantification of the peak amplitude (i.e the SR Ca 2+ content, CaSR), in WT (n=24) and YAP1-KO (n=22) CMs (B, right). Western blot analysis of SERCA2a and NCX1 protein levels (quantification on bottom) (C). Mean (± sem) I/V relations of NCX current (I NCX ) in WT (n=25) and YAP1-KO (n=24) CMs (D). Statistics of cell membrane capacitance (C m ) in WT (n=62) and YAP1-KO (n=66) CMs (E). Statistics: unpaired t test, * P <0.05.

Article Snippet: Plasmid containing full length YAP1 with T2A-mCherry tag (#74942, Addgene) was transfected together with envelope expressing plasmid 9 - PMS2.G (#12259, Addgene), and empty backbone packaging plasmid PSPAX2 (#12260, Addgene) into HEK293T cells using FuGENE® HD Transfection Reagent according to manufacturer’s instructions.

Techniques: Western Blot, Membrane

Journal: bioRxiv

Article Title: YAP1 Contributes to The Development of Contractile Force and Sarcomere Maturation in Human Pluripotent Stem Cell-Derived Cardiomyocytes

doi: 10.1101/2024.07.02.601803

Figure Lengend Snippet: Violin plot representation of nuclear translocation of YAP1 in hESC-WT cardiomyocytes in response to fibronectin (left) ( P <0.0001, N=3, n=145) and mechanical actuation (right) ( P <0.0001, N=3, n=502) (A). Violin plot representation of projected cell area in WT and YAP1-KO CMs in response to fibronectin ( P <0.001, N=3, n=253) (B) and mechanical actuation ( P <0.001, N=3, n=264) (C). Violin plot representation of sarcomere length in response to mechanical actuation in hESC-WT CMs ( P =0.04, N=3, n=122) (D). Attenuation of cellular response in presence of latrunculin A mediated cytoskeletal tension inhibition in response to fibronectin (E), mechanical actuation (F) in hESC-WT CMs. Representative confocal images of hESC-WT CMs stained for cardiac troponin-T (red) and DAPI (blue), images (E and F, left), quantification of projected cell area upon fibronectin deposition ( P <0.0001, N=3, n=215) (E, right) and mechanical actuation ( P <0.0001, N=3, n=328) (F, right). Statistics: Unpaired t test, ns: P >0.05, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Plasmid containing full length YAP1 with T2A-mCherry tag (#74942, Addgene) was transfected together with envelope expressing plasmid 9 - PMS2.G (#12259, Addgene), and empty backbone packaging plasmid PSPAX2 (#12260, Addgene) into HEK293T cells using FuGENE® HD Transfection Reagent according to manufacturer’s instructions.

Techniques: Translocation Assay, Inhibition, Staining

Journal: bioRxiv

Article Title: YAP1 Contributes to The Development of Contractile Force and Sarcomere Maturation in Human Pluripotent Stem Cell-Derived Cardiomyocytes

doi: 10.1101/2024.07.02.601803

Figure Lengend Snippet: Cardiomyocytes differentiated from WT and YAP1 deficient induced pluripotent stem cells (iPSCs) for 30 days were cast in 3D microtissues or kept in 2D adherent conditions for further ten days. Maturation associated changes in gene expression visualized as ratios left to right: myosin heavy chains isoforms (MYH7/MYH6), myosin light chains (MYL2/MYL7), and cardiac troponin I isoforms (TNNI3/TNNI1) expression in WT and YAP1-KO hIPSC-CMs at day 40 of differentiation in 2D and 3D cultures was quantified by RT-PCR (n=2) (A). Longitudinal sections of 3D microtissues stained at day 10 post casting for alpha-actinin (red) counterstained with DAPI (blue) (B). Analysis of sarcomere length at day 10 post casting ( P <0.0001, unpaired t test, n=63) (C). Cuore platform (Optics11 Life) was used to measure development of contraction force at different time points up to ten days following casting (D). Contractile force measurements at day 10 post casting ( P <0.01, unpaired t test, n=13) (E). Beating rate measurements up to 10 days post casting (F).

Article Snippet: Plasmid containing full length YAP1 with T2A-mCherry tag (#74942, Addgene) was transfected together with envelope expressing plasmid 9 - PMS2.G (#12259, Addgene), and empty backbone packaging plasmid PSPAX2 (#12260, Addgene) into HEK293T cells using FuGENE® HD Transfection Reagent according to manufacturer’s instructions.

Techniques: Gene Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining