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

Journal: Nature communications

Article Title: Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.

doi: 10.1038/s41467-022-35487-9

Figure Lengend Snippet: Fig. 4 | Ptk7 acts through noncanonical Wnt signaling. a Schematic repre- sentation of canonical and noncanonical Wnt signaling pathways. While canonical and noncanonical Wnt signaling pathways share components such as FZD and Dvl, other components such as LRP5/6 and β-Catenin are specific for canonical Wnt signaling. The figure was created with BioRender.com. b Representative images of organoids cultured in corresponding conditioned media with DMSO or inhibitors. Scale bar, 200 μm. c Quantification of data from (b). The percentage of cystic organoids in organoids cultured in conditioned media is shown. d Representative images of organoids treated with DMSO (carrier), Wnt3a or Wnt5a. Scale bar, 200 μm. e Quantification of data from (d). The percentage of cystic organoids in organoids cultured in conditioned media is shown. f Representative images of organoids cultured in Wnt5a with DMSO or inhibitors. Scale bar, 200 μm. g Quantification of data from (f). mean ± s.d. percentage of cystic organoids in organoids cultured in conditioned media. h Representative images of organoids

Article Snippet: Proteins used in ELISAs were obtained from R&D Systems: PTK7-Fc (R&D Systems, #9799-TK), biotinylated Wnt3a (R&D Systems, #BT1324), biotinylated Wnt5a (R&D Systems, #BT645).

Techniques: Protein-Protein interactions, Cell Culture

Journal: PLoS ONE

Article Title: Differential Palmit(e)oylation of Wnt1 on C93 and S224 Residues Has Overlapping and Distinct Consequences

doi: 10.1371/journal.pone.0026636

Figure Lengend Snippet: Panel A: HEK293T cells were transfected with 8XSuperTop/FopFlash , renilla luciferase, and indicated pcDNA expression constructs. DNA concentrations were held constant within each series of experiments. Values shown reflect the experimental data (Firefly/Renilla) minus FopFlash (Firefly/Renilla). The relative luciferase activity of wild-type Wnt1/Wnt3a was set at 1. Single and double Wnt1/Wnt3a mutants show significantly less activity than wild-type Wnts (p<1×10 −10 ). Differences between C93S and C93A mutants are not significant nor are those between S224A and C93A/S224A. Error bars indicate standard error for at least 15 data points from 3 independent replicates. Panel B: HEK293T cells were transfected as in Panel A, lysed, subjected to SDS-PAGE, and blotted onto PVDF. Blots were probed with anti-Wnt1 or anti-ß-tubulin. Panel C: The Western blots were scanned and analyzed using NIH ImageJ. This experiment was performed 3 times. Error bars represent +/− standard error. Panel D: LS/L cells were then transiently transfected with constructs encoding GFP or wild-type/mutant Wnt1. As before, the relative luciferase activity of wild-type Wnt1 was set at 1. Wnt1 and Wnt1C93A show statistically significant increases in reporter activity as compared to GFP (p<1×10 −7 ) while Wnt1S224A and Wnt1C93AS224A do not. Error bars represent standard error from at least 3 independent replicates with a total of 12 data points.

Article Snippet: Other materials include: TX-114 (Fisher); Lipofectamine 2000, Phalloidin-Alexa Fluor 633 (Invitrogen); Fugene HD (Promega); Mouse anti-GFP JL-8 (Clontech); FITC or Cy3 conjugated goat anti-rabbit IgG (H+L), Cy3 conjugated goat anti-mouse IgG IgM (H+L) and goat anti-mouse IgG (H+L)-AP, anti-rabbit DyL 649 (Jackson Laboratories); Dual Luciferase Reporter (Promega); anti-ß-tubulin, anti-PCNA (Santa Cruz Biotechnology); Dual Light System (Applied Biosystems); HEK293T cells (Developmental Studies Hybridoma Bank); anti-HA (Upstate); anti-Flotillin (BD Biosciences); anti-PDI (Calbiochem); anti-Giantin (Abcam); Optiprep (Accurate Chemicals); Tran 35 S label (MP Biomedicals); Protein A/G agarose beads, Sulfo-NHS-LC-biotin, NeutraAvidin Agarose (Pierce); En 3 Hance (Perkin Elmer); purified mouse Wnt3a (R&D Systems).

Techniques: Transfection, Luciferase, Expressing, Construct, Activity Assay, SDS Page, Western Blot, Mutagenesis

Journal: PLoS ONE

Article Title: Differential Palmit(e)oylation of Wnt1 on C93 and S224 Residues Has Overlapping and Distinct Consequences

doi: 10.1371/journal.pone.0026636

Figure Lengend Snippet: Panel A: Mock-transfected L cells were treated with control-conditioned media (Control-CM) or Wnt1/3a-conditioned media for 24 hrs. Note that Wnt1-CM was produced by standard L cells while Wnt3a was produced by E-cadherin transfected L cells (EL cells). Cells were then fixed, immunostained for PDI, and imaged with a confocal microscope. The length of the long axis of cells was measured using Adobe Photoshop (version 9.0.2). Error bars represent standard error. This experiment was repeated twice. At least 100 cells in 5 fields were measured. A Student's t-test shows that the difference in cell length between cells treated with control-CM and Wnt1- or Wnt3a-CM is statistically significant (p<0.005). Panel B: LS/L cells were treated with control-CM from mock-transfected L cells, Wnt1 conditioned medium, 33 mM NaCl, or 33 mM LiCl for 20–30 hrs and then assayed for activation of the SuperTopFlash reporter. Panel C: L cells were treated with 33 mM NaCl or LiCl for 24 hrs and then analyzed for cell elongation as in Panel A. Error bars represent the standard error from over 150 cells in two independent experiments. Neither NaCl nor LiCl has any significant effect on the length of the cells. Panel D: Purified mWnt3a was added to LS/L cells 24 hrs prior to conducting a dual luciferase assay to measure the activation of the SuperTopFlash construct. Error bars represent the standard error from 2 independent replicates with a total of 8 data points. Panel E: Mock-transfected L cells were incubated with purified mWnt3a for 24 hrs. Cells were then fixed, immunostained with anti-PDI, and imaged via confocal microscopy. The length of the long axis of individual cells was measured in Adobe Photoshop. Error bars represent the standard error from 2 independent replicates with a minimum of 107 cells measured for each data point.

Article Snippet: Other materials include: TX-114 (Fisher); Lipofectamine 2000, Phalloidin-Alexa Fluor 633 (Invitrogen); Fugene HD (Promega); Mouse anti-GFP JL-8 (Clontech); FITC or Cy3 conjugated goat anti-rabbit IgG (H+L), Cy3 conjugated goat anti-mouse IgG IgM (H+L) and goat anti-mouse IgG (H+L)-AP, anti-rabbit DyL 649 (Jackson Laboratories); Dual Luciferase Reporter (Promega); anti-ß-tubulin, anti-PCNA (Santa Cruz Biotechnology); Dual Light System (Applied Biosystems); HEK293T cells (Developmental Studies Hybridoma Bank); anti-HA (Upstate); anti-Flotillin (BD Biosciences); anti-PDI (Calbiochem); anti-Giantin (Abcam); Optiprep (Accurate Chemicals); Tran 35 S label (MP Biomedicals); Protein A/G agarose beads, Sulfo-NHS-LC-biotin, NeutraAvidin Agarose (Pierce); En 3 Hance (Perkin Elmer); purified mouse Wnt3a (R&D Systems).

Techniques: Transfection, Control, Produced, Microscopy, Activation Assay, Purification, Luciferase, Construct, Incubation, Confocal Microscopy