Journal: International Journal of Nanomedicine

Article Title: Second-generation aptamer-conjugated PSMA-targeted delivery system for prostate cancer therapy

doi: 10.2147/ijn.s23747

Figure Lengend Snippet: Figure 5 Bcl-2, Cyclin D1, and Wnt3a knock down following miRNA/PAMAM-PEG-APT complexes delivery to LNCaP cells. The cells were treated with miRNA/ PAMAM-PEG-APT (miRNA) and NC-miRNA/PAMAM-PEG-APT (NC-miRNA) complexes and processed for immunoblotting with anti-Bcl-2, anti-Cyclin D1, and anti-Wnt3a antibodies at 24, 48, and 72 hours post-transfection. β-actin was a loading control. Abbreviations: PAMAM, polyamidoamine; PEG, polyethylene glycol; APT, aptamer.

Article Snippet: The blotted membrane was immunostained with antibodies specific to anti-Bcl-2 (BioWorld, Visalia, CA), anti-Cyclin D1 (BioWorld) and anti-Wnt3a antibodies (R&D Systems Inc, Minneapolis, MN).

Techniques: Knockdown, Western Blot, Transfection, 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: eLife

Article Title: Anti-resonance in developmental signaling regulates cell fate decisions

doi: 10.7554/eLife.107794

Figure Lengend Snippet: ( A ) FACS data for Wnt I/O cell line with (+)/without (-) 24 hr of light exposure. Axes are of β-catenin (mCherry) v. TopFlash (APC-Cy5.5) ( B ) Live cell imaging of CRISPR tdmRuby3-β-cat, lentiviral 8X-TOPFlash-tdIRFP and DAPI, 16 hr after adding CHIR99021 (+CHIR) and Wnt3a (+Wnt3 a). ( C ) Quantifications of β-catenin and TopFlash fluorescence for no light, +16 hr light, +Wnt3 a, and +CHIR, where the mean of each condition was normalized to the mean of the no light condition and error bars represent SEM. ( D ) Flow for cell segmentation and heatmap generation from experimental data, using CellPose add TrackMate. From left to right: magenta (β-catenin) and cyan (TopFlash) images are passed into CellPose + Trackmate for segmentation and tracking. An example image of CellPose segmentation is shown under ‘segmented images,’ where the different colors correspond to the cell’s segmentation ID. Final images under ‘quantify and plot’ show quantification of β-catenin and TopFlash. ( E ) Mean fluorescent intensity (MFI) of β-catenin in the 24 hr light on condition, normalized to light off β-catenin. ( F ) MFI of TopFlash in the 24 hr light on condition, normalized to light off TopFlash. ( G ) Population mean MFI of β-catenin from live, single cell traces in indicated conditions, normalized to light off β-catenin. Initial drop in fluorescence is due to media bleaching. ( H ) Population mean MFI of TopFlash from live, single cell traces from indicated conditions, normalized to light off TopFlash. ( I ) Jitter plot of β-catenin mean nuclear fluorescent intensity (MFI) at the maximum intensity point in continuous light exposure conditions. Each point represents a single cell and the black line represents the mean of the population. ( J ) Jitter plot of TopFlash mean nuclear fluorescent intensity (MFI). Each point represents a single cell and the black line represents the mean of the population.

Article Snippet: HEK293T cells were treated using 10 μM of CHIR99201 (Stem Cell Technologies, 72052) or 2.5 nM of Wnt3a ligand (R&D Systems, 5036-WN) added into Dulbecco’s Modified Eagle Medium, high glucose GlutaMAX (Thermo Fisher Scientific, 10566016) medium supplemented with 10% fetal bovine serum (Atlas Biologicals, F-0500-D) and 1% penicillin-streptomycin.

Techniques: Live Cell Imaging, CRISPR, Fluorescence

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: JAK3 restrains inflammatory responses and protects against periodontal disease through Wnt3a signaling

doi: 10.1096/fj.201902697RR

Figure Lengend Snippet: Purified human monocytes were transfected with nontargeting control, Wnt3a, Dvl3, or GSK3β siRNAs, or a plasmid encoding β-catenin or a serine 9 to alanine constitutively active GSK3β mutant for 72 h and then stimulated with P. gingivalis over a 24 h time course in the absence or presence of a JAK3 inhibitor (1 nM T-1377). Cell-free supernatants and total cell lysates were harvested to determine the levels of TNFα, IL-6, and IL-12P40 and the transfection efficiency, respectively. (A to C) The silencing of Wnt3a or Dvl3 (A) significantly enhances the production of TNFα, IL-6, and IL-12P40 (B, C) in P. gingivalis-stimulated monocytes. The silencing of GSK3β or overexpression of β-catenin (D) abrogates the influence of JAK3 inhibition on the production of inflammatory cytokines and significantly reduces the levels of TNFα, IL-6, and IL-12P40 in P. gingivalis-stimulated monocytes (F). (G, H) Total cell lysates were probed with antibodies against β-catenin and the inserted HA tag to determine the transfection efficiency and its effect on the expression of β-catenin (G). (H) The production of TNFα, IL-6, and IL-12P40 in P. gingivalis-stimulated human monocytes overexpressing the GSK3β mutant. All the blots shown are representative of three to five experiments. For B, C, E, F, H, all the data were generated in the same experiment and represent the arithmetic mean±S.D. of three independent experiments. *, and *** indicate statistical significance at P<0.05 and P<0.001, respectively.

Article Snippet: Small interfering RNAs (siRNAs) against JAK3, NEDD4L, Wnt3a, GSK3β, and β-catenin were purchased from Dharmacon or Santa Cruz Biotechnology, Inc. All plasmids including pcDNA3-S33Y β-catenin (Cat.19286) with Flag tag, pcDNA3 Flag HA (Cat. 10792), which was constructed with HA and Flag tags, HA GSK3β S9A pcDNA3 (Cat. 14753), and pDONR223-JAK3 (Cat. 23944) were from Addgene.

Techniques: Purification, Transfection, Control, Plasmid Preparation, Mutagenesis, Over Expression, Inhibition, Expressing, Generated

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: JAK3 restrains inflammatory responses and protects against periodontal disease through Wnt3a signaling

doi: 10.1096/fj.201902697RR

Figure Lengend Snippet: Purified human monocytes were stimulated with P. gingivalis and total cell lysates were probed for the levels of Wnt3a with or without pretreatment of JAK3 inhibitor, T-1377 (1 nM) (A, B), or proteasome inhibitor, MG-132 (10 μM) (B, D). (C) The intensity ratio of Wnt3a to GAPDH was determined by densitometry. JAK3- specific siRNA was used to silence JAK3 in human monocytes (D, E) and THP-1 cells (F), and the total lysate of P. gingivalis-stimulated human monocytes was probed for the levels of immunoprecipitated Wnt3a and K48 ubiquitinated Wnt3a (D), the phosphorylation of Nedd4-2, and the levels of total Wnt3a and GAPDH (E, F). (F) JAK3 inhibitor (1nM T-1377) was also used to determine the effect of JAK3 on the phosphorylation of Nedd4-2 in P. gingivalis-stimulated THP-1 cells. (G to H) Purified human monocytes were transfected with nontargeting control siRNA or Nedd4-2-specific siRNA for 72 h and then stimulated with P. gingivalis over a 24 h time course. (G, H) Silencing of Nedd4-2 (G) significantly reduces the levels of TNFα, IL-6, and IL-12P40 in P. gingivalis-stimulated monocytes (H). All the blots shown are representative of three to five biological replicates. The data in (H) represent the arithmetic mean±S.D. of three independent experiments. *, and *** indicate statistical significance at P<0.05 and P<0.001, respectively.

Article Snippet: Small interfering RNAs (siRNAs) against JAK3, NEDD4L, Wnt3a, GSK3β, and β-catenin were purchased from Dharmacon or Santa Cruz Biotechnology, Inc. All plasmids including pcDNA3-S33Y β-catenin (Cat.19286) with Flag tag, pcDNA3 Flag HA (Cat. 10792), which was constructed with HA and Flag tags, HA GSK3β S9A pcDNA3 (Cat. 14753), and pDONR223-JAK3 (Cat. 23944) were from Addgene.

Techniques: Purification, Immunoprecipitation, Phospho-proteomics, Transfection, Control

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: JAK3 restrains inflammatory responses and protects against periodontal disease through Wnt3a signaling

doi: 10.1096/fj.201902697RR

Figure Lengend Snippet: Purified human monocytes were pretreated with T-1377 for 2 hours or transfected with nontargeting control, or JAK3-, Wnt3a- or Dvl3-specific siRNA for 72 h and then stimulated with P. gingivalis for the time indicated. Total cell lysates and nuclear lysates were harvested from separate groups. (A to D) Western blot of total cell lysates probed for the levels of phospho-NF-κB (Ser536) and GAPDH (D). (E, F) Ten micrograms of nuclear lysate were used to determine the transcription factor binding levels of NF-κB in human monocytes stimulated with P. gingivalis for 6 h. For A-D, the blots shown are representative of three to five experiments. For E and F, the data represent the arithmetic mean±S.D. of three independent experiments. *, and *** indicate statistical significance at P<0.05 and P<0.001, respectively.

Article Snippet: Small interfering RNAs (siRNAs) against JAK3, NEDD4L, Wnt3a, GSK3β, and β-catenin were purchased from Dharmacon or Santa Cruz Biotechnology, Inc. All plasmids including pcDNA3-S33Y β-catenin (Cat.19286) with Flag tag, pcDNA3 Flag HA (Cat. 10792), which was constructed with HA and Flag tags, HA GSK3β S9A pcDNA3 (Cat. 14753), and pDONR223-JAK3 (Cat. 23944) were from Addgene.

Techniques: Purification, Transfection, Control, Western Blot, Binding Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: JAK3 restrains inflammatory responses and protects against periodontal disease through Wnt3a signaling

doi: 10.1096/fj.201902697RR

Figure Lengend Snippet: Purified human monocytes (A to C) and THP-1 cells (D, E) were pretreated with a JAK3 inhibitor (1 nM T-1377) for 2 h and then stimulated with P. gingivalis (MOI of 10) for the indicated time. (A to E) Total cell lysates were probed for Wnt3a, Dvl3, p-GSK3β, p-β-catenin, and GAPDH (A, D) and mRNA levels of Wnt3a and Dvl3 were also detected after challenge with P. gingivalis for 2 h using qRT-PCR (C). (F to H) Western blot of human monocytes transfected with nontargeting or JAK3-specific siRNA for 72 h and then stimulated with P. gingivalis for the indicated time. Blots were probed with antibodies against Wnt3a, Dvl3, p-GSK3β, p-β-catenin, and GAPDH (G). The intensity ratio of each protein relative to that of GAPDH was determined by densitometry (B, E, F, H). (I, J) THP-1 cells were stimulated with Streptococcus sanguinis and Streptococcus gordonii (MOI of 10) for the indicated time. Total cell lysates were probed for p-JAK3, Wnt3a, Dvl3, and GAPDH. All the blots shown are representative of three to five biological replicates.

Article Snippet: Small interfering RNAs (siRNAs) against JAK3, NEDD4L, Wnt3a, GSK3β, and β-catenin were purchased from Dharmacon or Santa Cruz Biotechnology, Inc. All plasmids including pcDNA3-S33Y β-catenin (Cat.19286) with Flag tag, pcDNA3 Flag HA (Cat. 10792), which was constructed with HA and Flag tags, HA GSK3β S9A pcDNA3 (Cat. 14753), and pDONR223-JAK3 (Cat. 23944) were from Addgene.

Techniques: Purification, Quantitative RT-PCR, Western Blot, Transfection

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: JAK3 restrains inflammatory responses and protects against periodontal disease through Wnt3a signaling

doi: 10.1096/fj.201902697RR

Figure Lengend Snippet: (A, B) Eight- to 12-week-old C57BL/6 mice were divided randomly into one sham control group and two experimental groups (n=5 per group). The sham control group was treated with cellulose and 0.01% DMSO. The experimental groups were orally infected with P. gingivalis with or without pretreatment with the JAK3 inhibitor T-1377 (15 mg/kg) (A). Samples from the mouse oral cavity were examined by quantitative nested PCR to confirm P. gingivalis infection. (B) Representative electrophoresis images showing P. gingivalis DNA obtained from oral samples. (C, D) mRNA levels of TNFα, IL-6, and IL-12P40 (C), as well as Wnt3a and Dvl3 (D) were determined by RT-qPCR. (E) Total lysates of gingival tissue from upper jaws were probed for TNFα, IL-6, IL-12P40, and GAPDH. (F) Alveolar bone loss visualized by methylene blue/eosin staining and typical maxillae from sham-infected; P. gingivalis-infected; and T-1377-treated P. gingivalis-infected mice are presented. (G) Quantification of P. gingivalis-induced bone loss representing the distance from the CEJ to the ABC. Data are presented as the mean CEJ-ABC distance in mm±S.D.; n=5 mice per group. Error bars represent the S.D. *, and *** indicate statistical significance at P<0.05 and P<0.001, respectively. Data represent the arithmetic mean±S.D. of three independent experiments.

Article Snippet: Small interfering RNAs (siRNAs) against JAK3, NEDD4L, Wnt3a, GSK3β, and β-catenin were purchased from Dharmacon or Santa Cruz Biotechnology, Inc. All plasmids including pcDNA3-S33Y β-catenin (Cat.19286) with Flag tag, pcDNA3 Flag HA (Cat. 10792), which was constructed with HA and Flag tags, HA GSK3β S9A pcDNA3 (Cat. 14753), and pDONR223-JAK3 (Cat. 23944) were from Addgene.

Techniques: Control, Infection, Nested PCR, Electrophoresis, Quantitative RT-PCR, Staining

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: JAK3 restrains inflammatory responses and protects against periodontal disease through Wnt3a signaling

doi: 10.1096/fj.201902697RR

Figure Lengend Snippet: Immunohistochemistry staining of serial sections of gingival tissues from experimental mice treated with P. gingivalis, or P. gingivalis plus JAK3 inhibitor (T-1377, 15mg/kg), or sham control mice treated with DMSO and Cellulose with or without T-1377, showing the expression of Wnt3a (A to F) and Dvl3 (I to N). (G, O, H, P) The mean pixel intensity of Wnt3a (G) and Dvl3 (H), and the inflammatory cells infiltrated in the gingival tissue of P. gingivalis-infected mice (H, P) observed from more than 20 microscope fields of view are presented. Error bars represent the S.D. *, and *** indicate statistical significance at P<0.05 and P<0.001, respectively. Data represent the arithmetic mean±S.D. of three independent experiments.

Article Snippet: Small interfering RNAs (siRNAs) against JAK3, NEDD4L, Wnt3a, GSK3β, and β-catenin were purchased from Dharmacon or Santa Cruz Biotechnology, Inc. All plasmids including pcDNA3-S33Y β-catenin (Cat.19286) with Flag tag, pcDNA3 Flag HA (Cat. 10792), which was constructed with HA and Flag tags, HA GSK3β S9A pcDNA3 (Cat. 14753), and pDONR223-JAK3 (Cat. 23944) were from Addgene.

Techniques: Immunohistochemistry, Staining, Control, Expressing, Infection, Microscopy

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: JAK3 restrains inflammatory responses and protects against periodontal disease through Wnt3a signaling

doi: 10.1096/fj.201902697RR

Figure Lengend Snippet: (A) P. gingivalis infection activates proinflammatory signaling (not shown here) and thus induces the production of proinflammatory cytokines. Concurrently, JAK3 is phosphorylated upon the challenge of P. gingivalis and in turn phospho-inactivates Nedd4-2, leading to the decreased ubiquitination of Wnt3a and a subsequent increase in Wnt3a, Dvl3, and phospho-GSK3β levels, and the accumulation of β-catenin, which diminishes NF-κB signaling and ultimately constrains the proinflammatory immune response. In contrast, JAK3 inhibition promotes the activity of Nedd4-2, decreases the amount of Wnt3a, Dvl3, phospho-GSK3β, and β-catenin, which ultimately increases NF-κB activity, leading to increased proinflammatory cytokine production and inflammatory cell infiltration and the consequent exacerbation of inflammation-induced alveolar bone loss. (B) Briefly, activation of JAK3 is essential to restrain the production of pro-inflammatory cytokines (green) while inhibition of JAK3 leads to a robust increase of these cytokines (pink) in innate immune cells.

Article Snippet: Small interfering RNAs (siRNAs) against JAK3, NEDD4L, Wnt3a, GSK3β, and β-catenin were purchased from Dharmacon or Santa Cruz Biotechnology, Inc. All plasmids including pcDNA3-S33Y β-catenin (Cat.19286) with Flag tag, pcDNA3 Flag HA (Cat. 10792), which was constructed with HA and Flag tags, HA GSK3β S9A pcDNA3 (Cat. 14753), and pDONR223-JAK3 (Cat. 23944) were from Addgene.

Techniques: Infection, Ubiquitin Proteomics, Inhibition, Activity Assay, Activation 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) 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