Journal: PLoS ONE

Article Title: Distinct Transcriptional Networks in Quiescent Myoblasts: A Role for Wnt Signaling in Reversible vs. Irreversible Arrest

doi: 10.1371/journal.pone.0065097

Figure Lengend Snippet: Selected classes of genes up-regulated in quiescence.

Article Snippet: Control and treated MB were held in suspension for 48 hrs (with or without Wnt3a (R&D systems cat# 1324-WN) or sFRP2 (gift from Dr. Arun Dharmarajam, School of Anatomy & Human Biology The University of Western Australia), recovered from methocel, counted, re-suspended in GM without factors, plated at clonal density (400 cells/150 mm dish) and cultured for 7 days.

Techniques: Translocation Assay, RNA Binding Assay, Binding Assay, Derivative Assay, Histone Deacetylase Assay

Journal: PLoS ONE

Article Title: Distinct Transcriptional Networks in Quiescent Myoblasts: A Role for Wnt Signaling in Reversible vs. Irreversible Arrest

doi: 10.1371/journal.pone.0065097

Figure Lengend Snippet: (A) Exposure of adherent MB to rWnt3a (50 ng/ml) leads to β-cat nuclear localization, TOPflash activation and suppression of MyoD protein as compared to control cells. (B) rWnt 3a (50 ng/ml) does not enhance proliferation (BrdU incorporated in a 30′ pulse) in muscle cells: Asynchronous MB, G 0 MB, MB reactivated after synchronization in (R18) or differentiated myotubes (MT) [Note: all BrdU+ nuclei in myotube cultures were in residual mono-nucleated myobalsts]. Values represent the mean±SEM from three independent experiments. (C) Exogenous Wnt3a alters the quiescence program: Q-RTPCR analysis of control (blue bars) and Wnt-treated (pink bars) cells held in suspension for 48 hrs shows repression of MyoD and MyoG but induction of Myf5, indicating differential response of MRFs; repression of p21 and induction of CyclinD1 collectively suggesting a shift to a proliferative gene expression program; and finally, repression of quiescence-induced genes Rgs2 and Dkk3, consistent with this shift. Values represent the mean±SEM from three independent experiments. (D) Context-dependent response to Wnt enhancement. Cells in three different states (MB, G 0 or MT) were treated for 48 hours with 50ng/ml of rWnt3a. Of the MRFs, Myf5 mRNA is only induced by Wnt3a if the target cells are in G 0 . Values represent the mean±SEM from three independent experiments.

Article Snippet: Control and treated MB were held in suspension for 48 hrs (with or without Wnt3a (R&D systems cat# 1324-WN) or sFRP2 (gift from Dr. Arun Dharmarajam, School of Anatomy & Human Biology The University of Western Australia), recovered from methocel, counted, re-suspended in GM without factors, plated at clonal density (400 cells/150 mm dish) and cultured for 7 days.

Techniques: Activation Assay, Control, Reverse Transcription Polymerase Chain Reaction, Suspension, Gene Expression

Journal: PLoS ONE

Article Title: Distinct Transcriptional Networks in Quiescent Myoblasts: A Role for Wnt Signaling in Reversible vs. Irreversible Arrest

doi: 10.1371/journal.pone.0065097

Figure Lengend Snippet: (A) Wnt3A treatment of MB reduces clonogenic potential. Colony formation was measured after 48 hrs in control culture conditions (either in proliferating conditions-Mb, or in suspension culture-G 0 ), or in the presence of 50 ng/ml of rWnt3A. Cloning efficiency (a measure of self-renewal) was strongly reduced by Wnt3A supplementation and restored by simultaneous addition of 50ng/ml sFRP2. Values represent the mean±SEM from three independent experiments, p <0.05 (denoted by asterisk *). (B) Knockdown of Rgs2 and Dkk3 transcripts using siRNAs. siRNAs were designed against the putative Wnt regulators Rgs2, Dkk3 or an irrelevant gene (GAPDH) or a control scrambled siRNA sequence and transfected into C2C12 myoblasts along with a GFP plasmid. GFP + transfected cells were enriched by FACS, RNA isolated and analysed by Q-RT-PCR and the relative mRNA levels calculated. In each pair, the mRNA level is depicted of cells transfected with scrambled siRNA (blue bars) and cells transfected with the targeting siRNA (pink bars). Values represent the mean and SEM of 3 independent experiments. In each case, modest but reproducible reduction of the target transcript level is observed. (C) Reduction of Rgs2 and Dkk3 protein expression by siRNA-mediated knockdown. Western blot analysis of total protein isolated from control and knockdown C2C12 muscle cells probed with antibodies against Rgs2 (top) and Dkk3 (bottom). GAPDH protein levels indicate equal loading. Data depicted is representative of 3 independent experiments. (D) Rgs2 and Dkk3 expression is necessary for Wnt signaling. Knockdown of either Rgs2 or Dkk3 in growing or quiescent MB leads to suppression of TOPflash activity. Cells were treated and enriched as described in (B) and luciferase activity measured. Despite modest reduction of protein levels, strong reduction in TOPflash activity are seen, indicating a critical role for Rgs2 and Dkk3 in Wnt-βcat signaling. Values represent the mean and SEM of 3 independent experiments. (E,F) Knockdown cells (‘Rgs sh’ and ‘Dkk sh’) were enriched as described in (B) cultured in quiescence-inducing conditions, recovered from suspension culture and plated at clonogenic density for assessment of self-renewal (colony formation). Controls include untransfected cells (‘UT’) and control shRNA transfected cells (‘Con sh’). Typical plates with colony assays are shown in (E) and data are quantified as CFU (colony forming units) in (F). Values represent the mean and SEM of 3 independent experiments.

Article Snippet: Control and treated MB were held in suspension for 48 hrs (with or without Wnt3a (R&D systems cat# 1324-WN) or sFRP2 (gift from Dr. Arun Dharmarajam, School of Anatomy & Human Biology The University of Western Australia), recovered from methocel, counted, re-suspended in GM without factors, plated at clonal density (400 cells/150 mm dish) and cultured for 7 days.

Techniques: Control, Suspension, Cloning, Knockdown, Sequencing, Transfection, Plasmid Preparation, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Activity Assay, Luciferase, Cell Culture, shRNA