Journal: Advanced Science

Article Title: Aberrant Cholesterol Metabolism and Wnt/ β ‐Catenin Signaling Coalesce via Frizzled5 in Supporting Cancer Growth

doi: 10.1002/advs.202200750

Figure Lengend Snippet:

Article Snippet: L‐Wnt3a cell , ATCC , CRL‐2647.

Techniques: Staining, Western Blot, Sequencing, Control

Journal: Cancer Research Communications

Article Title: WNT4 Regulates Cellular Metabolism via Intracellular Activity at the Mitochondria in Breast and Gynecologic Cancers

doi: 10.1158/2767-9764.CRC-23-0275

Figure Lengend Snippet: BioID supports WNT4 localization to the mitochondria. A, Proteins enriched in HT1080 Wnt-BirA versus parental HT1080 cells lacking BirA construct expression. B, Overlap of proteins identified in HT1080 (A) versus HT1080-PKO and MM134 identifies n = 72 “high-confidence” WNT4-associated proteins. C, Gene ontology analysis for cellular compartment for WNT3A- versus WNT4-associated proteins. Dashed line = 1.3 ( P = 0.05). D, Network analysis of WNT3A- versus WNT4-associated proteins via subcell barcode. Enrichments against cell line HCC287 background shown; parallel results observed with other cell line background data, for example, MCF7. E, Proteins with predicted cytosolic or mitochondrial localization (subcell barcode) among “high-confidence” WNT4-associated proteins. Red = predicted mitochondrial localization, pink = mTOR complex in mitochondrial dynamics, biogenesis, and autophagy. F, Biotin treatment and streptavidin pulldown was performed as for MS studies, and candidate WNT4-associated proteins from E detected by immunoblotting. Total protein by Ponceau.

Article Snippet: Blots were probed with Streptavidin-HRP (Cell Signaling Technology #3999; RRID:AB_10830897) or antibodies used according to manufacturer's recommendations: WNT4 (R&D Systems, MAB4751; RRID:AB_2215448); WNT3A (Novus Biologicals, MAB13242); anti-HA-HRP conjugate (Cell Signaling Technology #2999; RRID:AB_1264166); DHRS2 (Sigma HPA053915; RRID:AB_2682307); mTOR (Cell Signaling Technology #2983; RRID:AB_2105622); STAT1 (Sigma HPA000931; RRID:AB_1080100).

Techniques: Construct, Expressing, Western Blot

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: Cell reports

Article Title: Six3 and Six6 Are Jointly Required for the Maintenance of Multipotent Retinal Progenitors through Both Positive and Negative Regulation

doi: 10.1016/j.celrep.2018.10.106

Figure Lengend Snippet: (A) RNA-seq not only confirmed most known DEGs but also identified additional DEGs, e.g., Fzd1 , Wnt3a , Cdon, and Wnt16 , in DKO retinas. Normalized counts generated using DESeq2 were used for plotting. The DEGs are in . (B) The gene set jointly regulated by Six3 and Six6 highly overlapped with the GRNs regulated by WNT3A, CTNNB1, POU4F2, or SOX2, which were among the top five upstream regulators in IPA. The predicted functions of DEGs matched the observed phenotypes. (C–N) Fzd1 (C–F), Wnt3a (G–J), and Wnt16 (K–N) were upregulated in E14.5 DKO retinas (F, J, and N) compared to those in Six3 F/F ; Six6 +/− retinas (C, G, and K), Six3 F/F ; Six6 −/− retinas (D, H, and L), and Six3 F/F ; α - Cre retinas (E, I, and M). Six3 deletion was shown in . Scale bar, 100 μm (C). n = 3 embryos

Article Snippet: Wnt3a (NM_009522) Mouse Tagged ORF Clone , Origene , Cat#MR227165.

Techniques: RNA Sequencing, Generated

Journal: Cell reports

Article Title: Six3 and Six6 Are Jointly Required for the Maintenance of Multipotent Retinal Progenitors through Both Positive and Negative Regulation

doi: 10.1016/j.celrep.2018.10.106

Figure Lengend Snippet: (A–D) CM marker Cdon was expanded in E14.5 DKO retinas (D) compared to that in Six3 F/F retinas (A), Six3 F/F ; α - Cre retinas (B), and Six3 F/F ; α - Cre ; Six6 +/− retinas (C). (E–H) Compared to vehicle controls (E), Wnt-3a (G) or GSK3β inhibitor CHIR99021 (H), but not Wnt-16b (F), expanded the size of CM (Cdon-positive Tubb3-negative) at the cost of NR identity at the periphery of mouse eyecups. (I–L) A working model. At E10.5–11.5, both Six3 and Six6 expression displayed a gradient along peripheral-central axis (I). In most-peripheral regions, low levels of Six3 and Six6 permitted Wnt/ β-catenin signaling, leading to CM specification (blue in (K), an illustration of Otx1 expression in E15.5 control retina in ). Moving centrally in the neuroretina, high levels of Six3 and Six6 protected multipotent NR progenitors directly or indirectly through (1) suppressing Wnt/β-catenin signaling and (2) maintaining the expression of retinogenic factors. When Six3 was deleted in E10.5 neuroretina using α - Cre in combination with Six6 deletion (J), the suppression of Wnt/β-catenin signaling was removed and the expression of retinogenic factors was disrupted. Retinal cells in far-peripheral regions gained CM identity at the expense of NR progenitors under the stimulation of Wnt/β-catenin signaling, whereas retinal cells in mid-peripheral regions maintained NR identity but lost multipotency (L, an illustration of Otx1 and Neurod1 expression in E15.5 DKO retinas in ). Scale bars, 100 μm (A), 50 μm (E). n = 3 retinas.

Article Snippet: Wnt3a (NM_009522) Mouse Tagged ORF Clone , Origene , Cat#MR227165.

Techniques: Marker, Expressing, Control

Journal: Cell reports

Article Title: Six3 and Six6 Are Jointly Required for the Maintenance of Multipotent Retinal Progenitors through Both Positive and Negative Regulation

doi: 10.1016/j.celrep.2018.10.106

Figure Lengend Snippet:

Article Snippet: Wnt3a (NM_009522) Mouse Tagged ORF Clone , Origene , Cat#MR227165.

Techniques: Recombinant, Plasmid Preparation, Software