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

Journal: Journal of neurochemistry

Article Title: Wnt-induced activation of glucose metabolism mediates the in vivo neuroprotective roles of Wnt signaling in Alzheimer disease

doi: 10.1111/jnc.14608

Figure Lengend Snippet: (A) Schematic of the protocol for assaying the correlation between Wnt3a signaling and glucose metabolism in the brain. After the indicated treatments, cognitive tests related to hippocampal function were performed: Large open field (B), NOR (C), NOL (D) and memory flexibility (E). Data obtained from APP/PS1 mice (under blue line). Data represent the mean ± SEM of n = 9 (number of animals), *p < 0.05; **p < 0.01, Bonferroni test.

Article Snippet: The neurons and slices were treated with recombinant Wnt3a (rWnt3a, 300 ng/mL, 0–24 h, cat: 1324-WN/CF, R&D Systems, USA), Aβ (5 μM), dickkopf-1 (Dkk1, 300 ng/mL, 0–24 h, antagonist of Wnt3a, as internal control, cat: 5897-DK/CF R&D System, USA), cytochalasin B (Cyt B, 1 μM, 3 h, inhibitor of GLUT transporters, this concentration is the IC 50 value, in the uptake we used 20 μM, Sigma-Aldrich USA), AZD5356 (20 nM, 12 h, an inhibitor of all Akt isoforms, cat: 5773, TOCRIS, UK), andrographolide (ANDRO, 50 μM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), carbonate of lithium (Li, 10 mM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), oligomycin (2 mM, inhibitor of ATP synthase, cat: 861944, Sigma-Aldrich USA) and 2-deoxy-D-glucose (2-DG, 7 mM, as a competitive inhibitor of hexokinase, Sigma-Aldrich USA).

Techniques:

Journal: Journal of neurochemistry

Article Title: Wnt-induced activation of glucose metabolism mediates the in vivo neuroprotective roles of Wnt signaling in Alzheimer disease

doi: 10.1111/jnc.14608

Figure Lengend Snippet: (A) Aβ oligomer structure determined by electron microscopy, treatment with Aβ reduced cell viability in a time-dependent manner, and the decrease in cell viability induced by Aβ was partially blocked by co-incubation with Wnt3a (arrow indicates apoptotic nucleus), n:3. (B) In contrast, co-incubation with Wnt3a+Aβ and Cyt B diminished the neuroprotective effect of the Wnt ligand. (C) Incubation with Aβ induced a dramatic decrease in 2-DG uptake. Wnt3a blocked this decrease, and Dkk partially inhibited the protective effect of Wnt3a. (D) The treatment of hippocampal neurons with ANDRO and Li stimulated the uptake of 2DG and this is inhibited by Cyt B. Data represent the mean ± SEM of n = 3 (independent experiments), each performed in triplicate. *p < 0.05; **p < 0.01, Bonferroni test.

Article Snippet: The neurons and slices were treated with recombinant Wnt3a (rWnt3a, 300 ng/mL, 0–24 h, cat: 1324-WN/CF, R&D Systems, USA), Aβ (5 μM), dickkopf-1 (Dkk1, 300 ng/mL, 0–24 h, antagonist of Wnt3a, as internal control, cat: 5897-DK/CF R&D System, USA), cytochalasin B (Cyt B, 1 μM, 3 h, inhibitor of GLUT transporters, this concentration is the IC 50 value, in the uptake we used 20 μM, Sigma-Aldrich USA), AZD5356 (20 nM, 12 h, an inhibitor of all Akt isoforms, cat: 5773, TOCRIS, UK), andrographolide (ANDRO, 50 μM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), carbonate of lithium (Li, 10 mM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), oligomycin (2 mM, inhibitor of ATP synthase, cat: 861944, Sigma-Aldrich USA) and 2-deoxy-D-glucose (2-DG, 7 mM, as a competitive inhibitor of hexokinase, Sigma-Aldrich USA).

Techniques: Electron Microscopy, Incubation

Journal: Journal of neurochemistry

Article Title: Wnt-induced activation of glucose metabolism mediates the in vivo neuroprotective roles of Wnt signaling in Alzheimer disease

doi: 10.1111/jnc.14608

Figure Lengend Snippet: The effects of the indicated treatments on the brain mRNA levels of genes encoding several proteins involved in glucose metabolism, including Campkiv (A), Cyclin D1 (B), c-Myc (C), which are known target genes of Wnt3a signaling. These target genes were increased by ANDRO and Li. The effect of treatments on the mRNA levels encoding additional metabolic proteins: Hk (D), Pk1 (E), Pfk1 (F), Akt (G) and Ampk (H). The mRNA levels of Hk, Pk1, Pfk1 and Akt increased after treatment with ANDRO. While the treatment with Li just recover the mRNA levels of Hk, Pkf1. Data from APP/PS1 mice (under blue line). Data represent the mean ± SEM of n = 3 (samples obtained from 3 different animals, by treatment), each performed in triplicate, *p < 0.05; **p < 0.01, Bonferroni test.

Article Snippet: The neurons and slices were treated with recombinant Wnt3a (rWnt3a, 300 ng/mL, 0–24 h, cat: 1324-WN/CF, R&D Systems, USA), Aβ (5 μM), dickkopf-1 (Dkk1, 300 ng/mL, 0–24 h, antagonist of Wnt3a, as internal control, cat: 5897-DK/CF R&D System, USA), cytochalasin B (Cyt B, 1 μM, 3 h, inhibitor of GLUT transporters, this concentration is the IC 50 value, in the uptake we used 20 μM, Sigma-Aldrich USA), AZD5356 (20 nM, 12 h, an inhibitor of all Akt isoforms, cat: 5773, TOCRIS, UK), andrographolide (ANDRO, 50 μM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), carbonate of lithium (Li, 10 mM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), oligomycin (2 mM, inhibitor of ATP synthase, cat: 861944, Sigma-Aldrich USA) and 2-deoxy-D-glucose (2-DG, 7 mM, as a competitive inhibitor of hexokinase, Sigma-Aldrich USA).

Techniques:

Journal: Journal of neurochemistry

Article Title: Wnt-induced activation of glucose metabolism mediates the in vivo neuroprotective roles of Wnt signaling in Alzheimer disease

doi: 10.1111/jnc.14608

Figure Lengend Snippet: (A) A time course of treatments showing that Aβ decreases the glycolytic rate in hippocampal neurons, in vitro. This effect was abolished by co-incubation with Wnt3a and this restoration was blocked by Dkk. (B) The activity of HK after 12 h of treatment with Aβ and/or the indicated compounds. (C) The activity of the PPP pathway after treatment with Aβ and the indicated compounds. This pathway remained unaltered. (D) ATP levels after Aβ treatment were dramatically reduced but rescued by co-treatment with Wnt3a. (E) The decrease in the levels of ATP correlated with the ATP/ADP ratio. Both ATP (E) and the ATP/ADP ratio increased in the presence of Wnt3a. Data represent the mean ± SEM of n = 3 (independent experiments), each performed in triplicate. *p < 0.05; **p < 0.01, Bonferroni test.

Article Snippet: The neurons and slices were treated with recombinant Wnt3a (rWnt3a, 300 ng/mL, 0–24 h, cat: 1324-WN/CF, R&D Systems, USA), Aβ (5 μM), dickkopf-1 (Dkk1, 300 ng/mL, 0–24 h, antagonist of Wnt3a, as internal control, cat: 5897-DK/CF R&D System, USA), cytochalasin B (Cyt B, 1 μM, 3 h, inhibitor of GLUT transporters, this concentration is the IC 50 value, in the uptake we used 20 μM, Sigma-Aldrich USA), AZD5356 (20 nM, 12 h, an inhibitor of all Akt isoforms, cat: 5773, TOCRIS, UK), andrographolide (ANDRO, 50 μM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), carbonate of lithium (Li, 10 mM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), oligomycin (2 mM, inhibitor of ATP synthase, cat: 861944, Sigma-Aldrich USA) and 2-deoxy-D-glucose (2-DG, 7 mM, as a competitive inhibitor of hexokinase, Sigma-Aldrich USA).

Techniques: In Vitro, Incubation, Activity Assay

Journal: Journal of neurochemistry

Article Title: Wnt-induced activation of glucose metabolism mediates the in vivo neuroprotective roles of Wnt signaling in Alzheimer disease

doi: 10.1111/jnc.14608

Figure Lengend Snippet: (A) The uptake of radioactive glucose in slices obtained from Wt and APP/PS1 mice (under blue line). Slices were treated with the indicated drugs for 1 h and then glucose uptake was measured. Treatment with Wnt3a and Wnt agonists increased the uptake of glucose in APP/PS1 slices and this was blocked by oligomycin. (B) The glycolytic rate after treatment with the indicated drugs. The decreased glycolytic rate of APP/PS1 slices was rescued by Wnt signaling. The activity of two key regulatory glycolytic enzymes, HK and PFK (C and D, respectively). The treatments, with the exception of rWnt3a affecting PFK, did not rescue the APP/PS1 mediated decreases in HK or PFK activity. Both ATP and the ATP/ADP ratio were decreased in slices from APP/PS1 mice, and both were increased in the presence of the agonists of Wnt3a signaling (E and F, respectively). Data represent the mean ± SEM of n = 3 (independent experiments), each performed in triplicate. *p < 0.05; **p < 0.01, Bonferroni test.

Article Snippet: The neurons and slices were treated with recombinant Wnt3a (rWnt3a, 300 ng/mL, 0–24 h, cat: 1324-WN/CF, R&D Systems, USA), Aβ (5 μM), dickkopf-1 (Dkk1, 300 ng/mL, 0–24 h, antagonist of Wnt3a, as internal control, cat: 5897-DK/CF R&D System, USA), cytochalasin B (Cyt B, 1 μM, 3 h, inhibitor of GLUT transporters, this concentration is the IC 50 value, in the uptake we used 20 μM, Sigma-Aldrich USA), AZD5356 (20 nM, 12 h, an inhibitor of all Akt isoforms, cat: 5773, TOCRIS, UK), andrographolide (ANDRO, 50 μM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), carbonate of lithium (Li, 10 mM, 12 h, agonist of Wnt3a signaling, Sigma-Aldrich USA), oligomycin (2 mM, inhibitor of ATP synthase, cat: 861944, Sigma-Aldrich USA) and 2-deoxy-D-glucose (2-DG, 7 mM, as a competitive inhibitor of hexokinase, Sigma-Aldrich USA).

Techniques: Activity Assay