pmscv ires egfp retroviral expression backbone  (Addgene inc)

Journal: Stem Cells (Dayton, Ohio)

Article Title: Common and Distinctive Functions of the Hippo Effectors Taz and Yap in Skeletal Muscle Stem Cell Function

doi: 10.1002/stem.2652

Figure Lengend Snippet: Taz controls satellite cell‐derived myoblast proliferation. (A): Representative images of 5‐ethyl‐2′‐deoxyuridine (EdU) incorporation in satellite cell‐derived myoblasts 48 hours following transduction with control retroviral vector (RV), or retroviral constructs encoding wild‐type human TAZ (TAZ RV) or constitutively active TAZ S89A (TAZ S89A RV), where green fluorescent protein (GFP) marks transduced cells. (B): Quantification of GFP+ cells with EdU incorporation shows that TAZ or TAZ S89A significantly increases EdU incorporation, and so proliferation rate ( n = 5 mice). (C): Representative images of EdU incorporation in satellite cell‐derived myoblasts following Yap and/or Taz small interfering RNA (Si)‐mediated knockdown using siControl, siYap, siTaz, or siYap + siTaz transfection. (D): Quantification of EdU incorporation shows that knockdown of Yap, Taz, or both Yap and Taz simultaneously reduces the proliferation rate ( n = 5 mice). (E): Relative gene expression measured by RT‐qPCR in satellite cell‐derived myoblasts compared with si Control under proliferation conditions following siControl, siYap, siTaz, or siYap + siTaz knockdown, normalized to Gapdh ( n = 5 mice except for myosin heavy chain/MyoD, where n = 3). Data presented as mean ± SEM, where an asterisk denotes significant difference from control RV/Si control ( p < .05) using Student's t test. Scale bar = 100 µm. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; EdU, 5‐ethyl‐2’‐deoxyuridine; GFP, green fluorescent protein; RV, retroviral vector; Si, small interfering RNA.

Article Snippet: Wild‐type (WT) TAZ, TAZ S89A, YAP S127A, or WT YAP was subcloned into a pMSCV‐IRES‐eGFP retroviral expression backbone (Addgene Plasmids 24809, 24815, 17791 and 17790) creating pMSCV‐3xFlag TAZ‐IRES‐eGFP and pMSCV‐3xFlag‐TAZ S89A‐IRES‐eGFP .

Techniques: Derivative Assay, Transduction, Control, Retroviral, Plasmid Preparation, Construct, Small Interfering RNA, Knockdown, Transfection, Gene Expression, Quantitative RT-PCR

Journal: Stem Cells (Dayton, Ohio)

Article Title: Common and Distinctive Functions of the Hippo Effectors Taz and Yap in Skeletal Muscle Stem Cell Function

doi: 10.1002/stem.2652

Figure Lengend Snippet: Taz regulates fusion of satellite cell‐derived myoblasts. (A, B): Satellite cells on isolated myofibers were transduced with retroviral vectors (RV) encoding wild‐type (WT) human TAZ (TAZ RV), constitutively active TAZ S89A (TAZ S89A RV) or Control RV, and cultured ex vivo for 72 hours, before being coimmunolabeled for eGFP and Pax7, or eGFP and Myogenin. Quantification of the proportion of (A) green fluorescent protein (GFP)/Pax7 and (B) GFP/myogenin satellite cells in the ex vivo cultures after 72 hours ( n = 3 mice). (C, D): Quantification of the proportion of expanded plated satellite cell‐derived myoblasts transduced with control RV, TAZ RV, or TAZ S89A RV and incubated in differentiation medium for 24 hours before being coimmunolabeled as myocytes for eGFP/Pax7 or eGFP/myogenin, illustrated in (E) ( n = 4 mice). (F): Representative images of expanded plated satellite cell‐derived myoblasts transduced with TAZ RV, TAZ S89A RV, or Control RV and incubated in differentiation medium before being coimmunolabeled for eGFP/myosin heavy chain (MyHC). (G): Quantification of the proportion of nuclei within GFP/MyHC myotubes shows that TAZ or TAZ S89A increases myogenic fusion ( n = 5 mice). (H): Representative images of satellite cell‐derived myoblasts transfected with small interfering RNA (siRNA) control or siRNA against Taz (SiRNA Taz) and immunolabeled for MyHC after 24 hours. (I): Quantification shows a trend toward reduced fusion ( p = .06) with siRNA Taz ( n = 3 mice). (J): Representative images of myotubes formed from satellite cell‐derived myoblasts isolated from WT ( Wwtr1 +/+ ) or Taz‐knockout ( Wwtr1 –/– ) mice after 24 hours in differentiation medium. (K): Quantification reveals less fusion in the Taz‐knockout ( Wwtr1 –/– ) mice compared with control WT ( n = 4 per genotype). Data are mean ± SEM, where an asterisk denotes significant difference ( p < .05) using a Student's t test from Control RV/siRNA control/ Wwtr1 +/+ as appropriate. Scale bars = 100µm. Abbreviations: GFP, green fluorescent protein; MyHC, myosin heavy chain; RV, retroviral vector; siRNA, small interfering RNA; WT, wildtype.

Article Snippet: Wild‐type (WT) TAZ, TAZ S89A, YAP S127A, or WT YAP was subcloned into a pMSCV‐IRES‐eGFP retroviral expression backbone (Addgene Plasmids 24809, 24815, 17791 and 17790) creating pMSCV‐3xFlag TAZ‐IRES‐eGFP and pMSCV‐3xFlag‐TAZ S89A‐IRES‐eGFP .

Techniques: Derivative Assay, Isolation, Transduction, Retroviral, Control, Cell Culture, Ex Vivo, Incubation, Transfection, Small Interfering RNA, Immunolabeling, Knock-Out, Plasmid Preparation

Journal: Stem Cells (Dayton, Ohio)

Article Title: Common and Distinctive Functions of the Hippo Effectors Taz and Yap in Skeletal Muscle Stem Cell Function

doi: 10.1002/stem.2652

Figure Lengend Snippet: TAZ operates through Tead4 to control myogenic differentiation. (A): Tead2–4 mRNA expression in regenerating tibialis anterior (TA) muscle collected at 1, 3, 5, 7, and 14 days post injury with cardiotoxin. Expression is presented as fold change compared with control undamaged TA ( n = 3). (B): Tead1–4 mRNA levels in C2C12 myoblasts at 24 and 48 hours in proliferation medium, and 24, 48, 72, and 96 hours in differentiation medium, expressed as fold change compared with 24 hours proliferation time‐point ( n = 3). (C): Representative Western blots of C2C12 myoblast lysate collected at 24 and 48 hours in proliferation medium and 24, 48, 72, and 96 hours in differentiation medium and immunoblotted for Yap, Taz, Tead1 (biological replicate 3), phosphorylated (P) Yap and Taz (biological replicate 2) and Tead4 (biological replicate 1) ( n = 4), with relevant α‐Tubulin or Gapdh loading controls. (D): Representative images of plated satellite cell‐derived myoblasts coimmunolabeled with Tead4 and Taz cultured under differentiation conditions for 24 hours. (E): Representative images of coimmunolabeling of myotubes formed from satellite cell‐derived myoblasts following TAZ S89A RV or Control RV transduction and/or small interfering RNA (siRNA) knockdown of Tead4 or siRNA Control. Green fluorescent protein (GFP) labels transduced cells and myosin heavy chain (MyHC) identifies myotubes. (F): Comparison of the proportion of nuclei in GFP+ve/MyHC+ve myotubes under each condition shows that TAZ S89A requires Tead4 to enhance differentiation ( n = 3 mice). (G): Representative images of coimmunolabeling of myocytes formed from satellite cell‐derived myoblasts following TAZ S89A RV or Control RV transduction and/or siRNA knockdown of Tead4 or siRNA Control, and 24 hours in differentiation medium. GFP labels transduced cells and Myogenin identifies myocytes. (H): Comparison of the proportion of GFP+ve/Myogenin+ve myocytes under each condition shows that TAZ‐induced myogenic differentiation operates through Tead4 ( n = 3 mice). Data are mean ± SEM from three mice, where an asterisk denotes significant difference ( p < .05) from Control RV/siRNA Control, or as indicated by bars, using a paired Student's t test. Scale bar = 100 µm. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; GFP, green fluorescent protein; MyHC, myosin heavy chain; P, Phosphorylated; RV, retroviral vector; siRNA, small interfering RNA; TA, tibialis anterior.

Article Snippet: Wild‐type (WT) TAZ, TAZ S89A, YAP S127A, or WT YAP was subcloned into a pMSCV‐IRES‐eGFP retroviral expression backbone (Addgene Plasmids 24809, 24815, 17791 and 17790) creating pMSCV‐3xFlag TAZ‐IRES‐eGFP and pMSCV‐3xFlag‐TAZ S89A‐IRES‐eGFP .

Techniques: Control, Expressing, Western Blot, Derivative Assay, Cell Culture, Transduction, Small Interfering RNA, Knockdown, Comparison, Retroviral, Plasmid Preparation