Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Quantification of change in cell density (number of DAPI positive nuclei per cm 2 imaged area) upon FOXM1 overexpression or knockdown, 72h post transfection. Data is normalized to appropriate controls (Empty vector for pFOXM1 and non-targeting siRNA for siFOXM1). Bars represent Mean + SD (n = 4). P<0.0001 (Student’s t-test). B. Heatmap showing changes in gene expression of a panel of representative markers over a timecourse of RPE culture where cells are seeded at high (100000 cells/cm 2 ) or low (8000 cells/cm 2 ) density. C. Plot showing differential expression of BMP7 and Wnt5B transcripts extrapolated from the microarray data. The shaded area represents 95% confidence intervals around the point estimates (circles) of the difference between the mean high density expression vs the mean low density expression.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Over Expression, Knockdown, Transfection, Plasmid Preparation, Gene Expression, Quantitative Proteomics, Microarray, Expressing

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: A. Immunocytochemistry for PMEL17 where cells are seeded at either low density (16000 cells/cm 2 ) in the presence or absence of BMP4/7 (top left) or at high density (25000 cells/cm 2 ) in the presence or absence of Wnt5B (bottom left) and cultured for a period of 14 days. Also shown is the expression of BEST1 under the same conditions (top and bottom right). ACTB and B2M are used as housekeeping genes. Bars represent Mean + SD (n = 3). B. Quantification of immunocytochemistry for % CRALBP at Day 21 where cells are either treated with media alone (Control) or media supplemented with 10μM LDN-193189 added at Day 2,4,6,8,11,14 or 18. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). C. Quantification of immunocytochemistry for % CRALBP at Day 28 where cells are either treated with media alone (Control) or media supplemented with 10μM WAY-262611 added at Day 2,7,14 or 21. * indicates significant difference between control and compound treatment (One way ANOVA with Dunnett’s multiple comparisons). D. qPCR based measurement of BMP7 and Wnt5B transcript expression at Day 10 post siFOXM1 transfection (relative to transfection with non-targeting siRNA used as a control). GAPDH , HPRT1 and IPO8 were used as housekeeping genes. Bars represent Mean + SD (n = 3). P<0.05 (Student’s t-test).

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Immunocytochemistry, Cell Culture, Expressing, Control, Transfection

Journal: PLoS ONE

Article Title: A FOXM1 Dependent Mesenchymal-Epithelial Transition in Retinal Pigment Epithelium Cells

doi: 10.1371/journal.pone.0130379

Figure Lengend Snippet: RPE first acquire a mesenchymal morphology upon dissociation and culture followed by proliferation and mesenchymal-epithelial transition to re-uptake an epithelial phenotype. Proliferation of RPE is directly regulated by FOXM1 which also affects expression of BMP7 and Wnt5B by an unknown mechanism. Both these activities are required for successful MET and epithelialization.

Article Snippet: Where required, media was supplemented with recombinant human Wnt5B (500ng/ml; R&D Systems), BMP-4/7 (75ng/ml; R&D Systems), Thiostrepton (Sigma), LDN-193189 (10μM; Stemgent), WAY-262611 (10μM; Enzo Lifesciences).

Techniques: Expressing

Journal: Molecular cancer research : MCR

Article Title: Cholesterol Esterification Inhibition Suppresses Prostate Cancer Metastasis by Impairing the Wnt/β-catenin Pathway

doi: 10.1158/1541-7786.MCR-17-0665

Figure Lengend Snippet: (A) Immunofluorescent staining of Wnt3a in DU145 PTEN-KD treated with avasimibe (10 μM, 2 days) or IWP-2 (10 μM, 1 day). Arrows indicate membrane localized Wnt3a. Scale bar: 25 μm. Insert scale bar: 10 μm. (B) Quantification of cells with membrane Wnt3a after avasimibe or IWP-2 treatment as indicated in (A). (C) Immunoblot of antibodies against Wnt3a and β-actin in DU145 PTEN-KD treated with avasimibe (10 μM, 2 days). Medium Wnt3a was immunoprecipitated and normalized by precipitated protein concentration. Error bars represent SD (n = 3). *p < 0.05. (D) Representative images of migration of cells pre-treated with avasimibe (10 μM, 2 days) and subsequent Wnt3a supplement (100 ng/mL). Scale bar: 50 μm. (E) Quantitation of migrated cells shown in (D). Error bars represent SD (n = 3). ***p < 0.0005. (F) Schematic showing the molecular mechanism. In metastatic PCa, high level of lipid synthesis and/or uptake provides fatty acid substrates for Wnt acylation. This process is accompanied by conversion of excess cholesterol to CE by ACAT and stored in lipid droplets. Wnt acylation allows secretion and binding of Wnt to the membrane to drive cell migration and invasion. Suppressing cholesterol esterification by ACAT inhibition downregulates SREBP through negative feedback loop. As a result, lipogenesis pathway is suppressed, which limits the availability of free fatty acids required for Wnt acylation. Metastasis is then inhibited by reduced secretion of Wnt protein. SFA: saturated fatty acid, MUFA: unsaturated fatty acid, LD: lipid droplet.

Article Snippet: Recombinant human Wnt3a was purchased from Bio-Techne Corporation.

Techniques: Staining, Membrane, Western Blot, Immunoprecipitation, Protein Concentration, Migration, Quantitation Assay, Binding Assay, Inhibition

Journal: Scientific Reports

Article Title: Coordination of WNT signaling and ciliogenesis during odontogenesis by piezo type mechanosensitive ion channel component 1

doi: 10.1038/s41598-019-51381-9

Figure Lengend Snippet: Treatment of Yoda1 upregulates expression of WNT16 gene which promotes mineralization of SHED. ( a , b ) Expression of WNT and DACT3 genes. SHED were cultured with or without 5 μM Yoda1 for 24 hrs. Total RNA was prepared from the cells and analyzed by real-time RT-PCR for WNT expression ( a ) and DACT3 expression ( b ). ( c ) Alizarin Red S staining after WNT16 treatment to SHED. After 7 days of culture of SHED in odontogenic induction media in the presence or absence of 300 ng/ml exogenous WNT16, Alizarin Red staining was performed in SHED. The Alizarin Red-positive areas were quantified using ImageJ. Scale bar, 150 μm. The data, as shown, are representative of three independent experiments with similar results, and error bars indicate standard deviations. Statistical analysis was performed using analysis of variance (** p < 0.01).

Article Snippet: Yoda1 and XAV939 were purchased from Tocris Bioscience (Bristol, UK) and recombinant human WNT16 protein from R&D systems (Minneapolis, USA).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Staining

Journal: Blood

Article Title: Inhibition of WNT signaling in the bone marrow niche prevents the development of MDS in the Apc del/ + MDS mouse model

doi: 10.1182/blood-2016-08-736454

Figure Lengend Snippet: Loss of 1 copy of Ctnnb1 in an Apc-haploinsufficient microenvironment prevents or delays the development of MDS by 8 to 10 months. (A) Kaplan-Meier survival curves for Apcdel/+ (A; n = 4), Apcdel/+, Ctnnb1del/+ (AC; n = 11), Ctnnb1del/+ (C; n = 3), and Apcfl/+, Ctnnb1fl/+ (Cre−; n = 9) recipient mice. Median survival of Apcdel/+ and Apcdel/+, Ctnnb1del/+ recipient mice was significantly different (115 vs 413 days; P < .0001). All control mice (C and Cre−) survived until the end of the study, with the exception of 1 Apcfl/+, Ctnnb1fl/+ recipient that died at 343 days, likely due to a hemorrhagic renal cyst. (B) Percentage of CD71+Ter119+ erythroid cells, Gr1+CD11b+ myeloid cells, and CD19+IgM+ B cells in spleen isolated from Cre− (∼400 days), A (70-115 days), and AC (303-413 days) recipients that eventually displayed a fatal anemia. At sacrifice, the AC cell populations were more similar to A than Cre− recipients. (C) RBC and Hb counts in all 4 cohorts over time. The development of anemia is delayed in AC recipients after 35 weeks (a point in time when all A recipients have already been sacrificed due to severe anemia). In Cre− control recipients, 2 mice developed moderate anemia at 57 weeks. However, 1 mouse had an apparent colorectal tumor, and the other had a hemorrhagic renal cyst; neither had developed MDS. (D) MSCs were isolated from Cre− (control), A, and AC littermates 2 months posttreatment with pIpC to induce Cre-mediated deletion, and before development of disease. Following in vitro Wnt3a stimulation for 6 hours, nuclear and cytoplasmic fractions were isolated and immunoblotted with Ctnnb1, β-actin (cytoplasmic), and Hdac1 (nuclear) antibodies. Quantification of 3 independent experiments shows increased nuclear and cytoplasmic Ctnnb1 protein expression in Apcdel/+ MSCs that is reduced by ∼50% upon haploinsufficient loss of Ctnnb1. (E) RNA was isolated from Cre−, A, and AC MSCs (no Wnt3a stimulation), transcribed to complementary DNA (cDNA), and PCRs (run in triplicate) were quantified using Fast-SYBR Green. Gene expression was normalized to Gapdh and data are presented as mean ± standard error of the mean (SEM) of 3 independent experiments. ***P < .0001, **P < .001, *P < .05. NS, not significant.

Article Snippet: MSCs were stimulated with 50 ng/mL recombinant mouse Wnt3a (Pepro Tech, Rocky Hill, NJ) or human WNT3A (R&D Systems, Minneapolis, MN) with or without 50 nM PT for times indicated.

Techniques: Isolation, In Vitro, Expressing, SYBR Green Assay

Journal: Blood

Article Title: Inhibition of WNT signaling in the bone marrow niche prevents the development of MDS in the Apc del/ + MDS mouse model

doi: 10.1182/blood-2016-08-736454

Figure Lengend Snippet: Pyrvinium modulation of WNT signaling is more effective before the onset of moderate-severe anemia. (A) Two-month-old Mx1-Cre−Apcfl/+ (Cre−) or Mx1-Cre+Apcfl/+ (Cre+, also referred to as Apcdel/+) recipients were treated with pIpC (to induce Apc deletion) and vehicle (DMSO) or PT 2 weeks before lethal irradiation and transplantation with WT (CD45.1) BM cells. Mice were injected twice per week with 0.01, 0.1, or 0.5 mg/kg PT or DMSO until sacrifice. Kaplan-Meier curves for overall survival show that mice treated with 0.1 mg/kg or 0.5 mg/kg PT survived about 1 to 2 months longer than vehicle-treated mice (P = .0302 and P = .0064, respectively). (B) RBC and Hb counts of DMSO and PT-treated mice at ∼100 days posttransplant (for some mice in the DMSO and 0.01 mg/kg PT groups, counts from <100 days were plotted since they died before 100 days). The RBC and Hb counts of 0.5 mg/kg PT-treated mice were higher than 0.01 mg/kg PT-treated mice, indicating the administration of 0.5 mg/kg PT delays development of anemia (P = .0018 and P = .0320). The 2 mice in the 0.1 mg/kg PT-treated group that survived beyond 200 days had noticeably higher RBC and Hb counts at 100 days (circled). (C) Cre+ recipients were treated with 0.5 mg/kg PT once they developed mild (Hb, 12-13.5 g/dL), moderate (Hb, 10-11.5 g/dL), or severe anemia (Hb, <10 g/dL). A Kaplan-Meier survival curve of all PT-treated mice indicates survival is extended by almost 2 months (DMSO vs PT: 104 days vs 159 days; P < .0001). (D) The average median survival of recipients from the mild, moderate, and severe anemia group vs the DMSO-treated control group is shown. A longer survival is achieved if treatment is started before the onset of severe anemia. (E) MSCs were isolated from Apcdel/+mice ∼2 months post-pIpC-induced deletion and before development of disease. MSCs were treated with or without 50 ng/mL Wnt3a ± 50 nM PT, as indicated, for 16 hours. A representative immunoblot of nuclear and cytoplasmic fractions immunoblotted with Ctnnb1, β-actin (cytoplasmic), and Hdac1 (nuclear) is shown. In 3 independent experiments, PT treatment decreased Wnt3a-mediated elevation of Ctnnb1 by 56% ± 11.5% (P = .04). (F) Apcdel/+ MSCs were stimulated in vitro ± 50 ng/mL Wnt3a ± 50 nM PT for 16 hours. RNA was isolated and transcribed to cDNA, and PCRs (run in triplicate) were quantified using Fast-SYBR Green. Gene expression was normalized to Gapdh and data are presented as mean ± SEM of 3 independent experiments. **P < .001, *P < .05. NR, not reached.

Article Snippet: MSCs were stimulated with 50 ng/mL recombinant mouse Wnt3a (Pepro Tech, Rocky Hill, NJ) or human WNT3A (R&D Systems, Minneapolis, MN) with or without 50 nM PT for times indicated.

Techniques: Irradiation, Transplantation Assay, Injection, Isolation, Western Blot, In Vitro, SYBR Green Assay, Expressing

Journal: Blood

Article Title: Inhibition of WNT signaling in the bone marrow niche prevents the development of MDS in the Apc del/ + MDS mouse model

doi: 10.1182/blood-2016-08-736454

Figure Lengend Snippet: Pyrvinium suppresses WNT activation in MSCs isolated from patients with myeloid neoplasms with a del(5q). (A) MSCs were isolated from the BM of 2 del(5q) patients with primary MDS or t-MDS and were treated in vitro with or without 50 ng/mL WNT3A ± 50 nM PT, as indicated, for 16 hours. Nuclear and cytoplasmic fractions were isolated and immunoblotted with antibodies specific for CTNNB1, β-actin, and Lamin A/C. Quantitation of the immunoblots revealed a ∼50% reduction in CTNNB1 levels in samples treated with PT. *A smaller CTNNB1 degradation product. (B) Patient MSCs were treated with WNT3A ± 50 nM PT for 16 hours. RNA was isolated and transcribed to cDNA, and PCRs (run in triplicate) were quantified using Fast-SYBR Green. Gene expression was normalized to the ACTB gene and data are presented as mean ± SEM of 1 patient sample, run in triplicate. PT significantly decreased WNT3A-mediated transcription of WNT target genes, but not the control GAPDH gene. *P < .05. Increased GAPDH may reflect emerging evidence suggesting that GAPDH gene expression can be modulated by external factors.58

Article Snippet: MSCs were stimulated with 50 ng/mL recombinant mouse Wnt3a (Pepro Tech, Rocky Hill, NJ) or human WNT3A (R&D Systems, Minneapolis, MN) with or without 50 nM PT for times indicated.

Techniques: Activation Assay, Isolation, In Vitro, Quantitation Assay, Western Blot, SYBR Green Assay, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: OGD leads to progressive injury in neurons and reduces endogenous Wnt1 expression over time. In A, primary hippocampal neurons were exposed to progressive durations of OGD at 1, 2, 3 and 4 hours and neuronal survival was determined 24 hours later by trypan blue dye exclusion assay. Neuronal survival was decreased to 73 ± 3% (1 hour), 51 ± 4% (2 hours), 32 ± 3% (3 hours), and 20 ± 3% (4 hours) following OGD exposure when compared with untreated control cultures (86 ± 3%, *p < 0.01 vs. Control). Each data point represents the mean and SEM from 6 experiments. In B, neuronal protein extracts (50 µg/lane) were immunoblotted with anti-Wnt1 (Wnt1) at 1, 6 and 24 hours following OGD exposure. Wnt1 expression is initially elevated at 1 hour and 6 hours, but then progressively and significantly is reduced at 24 hours following OGD exposure (*p < 0.01 vs. control)

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Expressing, Exclusion Assay, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Transient transfection of Wnt1 increases neuronal survival and prevents genomic DNA degradation and membrane ps externalization. In A, overexpression of Wnt1 was performed under the control of a CMV promoter with Wnt1 cDNA and cell survival was determined 24 hours after OGD exposure through the trypan blue dye exclusion method. Representative images illustrate decreased trypan blue staining during Wnt1 transient transfection. Significant cell injury and trypan blue staining occurs during OGD alone in wildtype cells and during vector transfection. Quantification of data demonstrates that OGD significantly decreased percent cell survival when compared to the control cells. In contrast, Wnt1 significantly increased cell survival (*p < 0.01 vs. OGD). Each data point represents the mean and SEM from six experiments. Control = untreated neurons. In B, overexpression of Wnt1 was performed under the control of a CMV promoter with Wnt1 cDNA and genomic DNA degradation was determined 24 hours after OGD exposure through TUNEL. Representative images illustrate decreased TUNEL staining during Wnt1 transient transfection. Significant DNA fragmentation occurs during OGD alone in wildtype cells and during vector transfection. Quantification of data demonstrates that OGD results in marked DNA fragmentation when compared to the control cells. In contrast, Wnt1 significantly prevents DNA fragmentation (*p < 0.01 vs. OGD). Each data point represents the mean and SEM from six experiments. Control = untreated neurons. In C, overexpression of Wnt1 was performed under the control of a CMV promoter with Wnt1 cDNA and membrane PS externalization was determined 24 hours after OGD exposure through annexin V phycoerythrin (green fluorescence). Representative images illustrate decreased PS staining during Wnt1 transient transfection. Significant membrane PS externalization occurs during OGD alone in wildtype cells and during vector transfection. Quantification of data demonstrates that OGD results in marked PS exposure when compared to the control cells. In contrast, Wnt1 significantly prevents PS externalization (*p < 0.01 vs. OGD). Each data point represents the mean and SEM from six experiments. Control, untreated neurons.

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Transfection, Membrane, Over Expression, Control, Staining, Plasmid Preparation, TUNEL Assay, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Wnt1 signaling is necessary for neuronal protection against OGD. In A, increasing concentrations of Wnt1 protein result in significantly increased neuronal survival assessed by trypan blue exclusion 24 hours after OGD (*p < 0.01 vs. OGD). In B, increasing concentrations of an antibody to Wnt1 (Wnt1Ab) during OGD do not alter neuronal survival assessed by trypan blue exclusion 24 hours after OGD when compared to neurons exposed to OGD alone. In C, increasing concentrations of an antibody to Wnt1 (Wnt1Ab) applied with Wnt1 (100 ng/ml) resulted in progressive loss of Wnt1 protection and increased neuronal cell injury assessed by trypan blue staining 24 hours after OGD (*p < 0.01 vs. OGD). In D, inhibition of Wnt1 signaling with DKK-1 (0.5 µg/ml), an antagonist of the Wnt/β-catenin pathway, administered with Wnt1 (100 ng/ml) significantly reduces protection by Wnt1 and neuronal cell survival assessed by trypan blue staining 24 hours after OGD (*p < 0.01 vs. OGD; †p < 0.01 vs. Wnt1/OGD). In all cases control = untreated neurons. Each data point represents the mean and SEM from six experiments.

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Staining, Inhibition, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Wnt1 relies upon the PI 3-K pathway and Akt1 to provide neuronal protection. In A, primary neuronal protein extracts (50 µ/lane) were immunoblotted with anti-phosphorylated-Akt1 (p-Akt1, Ser 473 ) or anti-total Akt1 at 6 hours following OGD. Application of Wnt1 (100 ng/ml) in untreated wildtype neurons or in the presence of OGD significantly elevated p-Akt1 expression to a greater extent than OGD alone. This increased expression of p-Akt1 by Wnt1 was blocked by the PI 3-K inhibitor wortmannin (0.5 µM) and by the specific Akt1 inhibitor SH-5 (20 µM). Total Akt1 is not altered (*p < 0.01, vs. OGD; †p < 0.01 vs. Wnt1/OGD). Each data point represents the mean and SEM from six experiments. In B, primary neurons treated with Wnt1 (100 ng/ml) increased neuronal survival assessed by trypan blue staining 24 hours after OGD. Yet, application of wortmannin (0.5 µM) or SH-5 (20 µM) at concentrations that block activation of Akt1 activation significantly reduced protection by Wnt1 24 hours after OGD (*p < 0.01, vs. OGD; †p < 0.01 vs. Wnt1/OGD). Each data point represents the mean and SEM from six experiments.

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Expressing, Staining, Blocking Assay, Activation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Wnt1 uses Akt1 to block apoptotic membrane PS exposure and genomic DNA degradation during OGD. In A, representative images illustrate that recombinant human Wnt1 protein (100 ng/ml) significantly blocks neuronal genomic DNA degradation assessed by TUNEL and membrane PS externalization assessed by annexin V phycoerythrin (green fluorescence) 24 hours following OGD. In contrast, inhibition of Wnt1 (100 ng/ml) signaling with Wnt1Ab (1 µg/ml) or with application of DKK-1 (0.5 µg/ml) or inhibition of Akt1 with SH-5 (20 µM) during Wnt1 application leads to the loss of Wnt1 protection. In B, quantification of data illustrates that DNA fragmentation and membrane PS externalization were significantly increased 24 hours following OGD when compared to untreated neuronal control cultures during Wnt1 (100 ng/ml) application with Wnt1Ab (1 µg/ml), DKK-1 (0.5 µg/ml), or SH-5 (20 µM). Each data point represents the mean and SEM from six experiments.

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Blocking Assay, Membrane, Recombinant, TUNEL Assay, Fluorescence, Inhibition, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Wnt1 inhibits mitochondrial depolarization and prevents the release of cytochrome c during OGD. In A, OGD leads to a significant decrease in the red/green fluorescence intensity ratio of mitochondria using the cationic membrane potential indicator JC-1 within 3 hours when compared with untreated control neurons, demonstrating that OGD leads to mitochondrial membrane depolarization. Application of Wnt1 (100 ng/ml) during OGD significantly increased the red/green fluorescence intensity of mitochondria in neurons, illustrating that mitochondrial membrane potential was restored by Wnt1. In contrast, inhibition of Wnt1 (100 ng/ml) signaling with Wnt1Ab (1 µg/ml) or with application of DKK-1 (0.5 µg/ml) or inhibition of Akt1 with SH-5 (20 µM) during Wnt1 application and OGD resulted in mitochondrial depolarization similar to OGD exposure alone. In B, the relative ratio of red/green fluorescent intensity of mitochondrial staining in untreated control neurons, in neurons exposed to OGD, during Wnt1 (100 ng/ml)/OGD application alone or during Wnt1 (100 ng/ml)/OGD with Wnt1Ab (1 µg/ml), DKK-1 (0.5 µg/ml), or SH-5 (20 µM) was measured in six independent experiments with analysis performed using the public domain NIH Image program ( http://rsb.info.nih.gov/nih-image ) (Wnt1/OGD vs. OGD, *p < 0.01; Wnt1/OGD with Wnt1Ab, DKK-1, or SH-5 vs. Wnt1/OGD, †p < 0.01). In C and D, equal amounts of mitochondrial (mito) or cytosol (cyto) protein extracts (50 µg/lane) were immunoblotted demonstrating that Wnt1 (100 ng/ml) significantly prevented cytochrome c release from mitochondria within 3 hours after OGD but Wnt1Ab (1 µg/ml), DKK-1 (0.5 µg/ml), or SH-5 (20 µM) during Wnt1/OGD application prevented Wnt1 from maintaining cytochrome c in the mitochondria (Wnt1/OGD vs. OGD, *p < 0.01; Wnt1/OGD with Wnt1Ab, DKK-1, or SH-5 vs. Wnt1/OGD, †p < 0.01).

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Fluorescence, Membrane, Control, Inhibition, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Transient cerebral ischemia blocks endogenous Wnt1 cortical expression but exogenous Wnt1 is protective against cerebral ischemia. In A and B, focal cerebral ischemia was induced by insertion of a monofilament thread (4-0) into the internal carotid artery and blockade of the origin of MCA. Reperfusion was performed following 90 minutes ischemia by withdrawal of the thread. For A, cortical protein extracts (50 µg/lane) were immunoblotted with anti-Wnt1 (Wnt1) at 1, 6 and 24 hours following OGD exposure. Similar to the effects upon Wnt1 expression with OGD in neuronal cultures, Wnt1 expression is initially elevated at 1 hour and 6 hours, but then progressively and significantly is reduced at 24 hours following OGD exposure (*p < 0.01 vs. control). Wnt1 expression on the contralateral non-infarction hemisphere was not altered from control, illustrating that the generation of cerebral ischemia directly led to changes in endogenous Wnt1 expression. In B, Wnt1 protein (24 µg/kg) was injected into the internal carotid artery through the external carotid artery at 30 minutes prior to the onset of MCAO and at the onset of reperfusion. Animals were euthanized 24 hours following ischemia and the infarct size was determined by 2,3,5-triphenytetrazolium (TTC) staining. Representative images show that the infarct size (white in color) was significantly reduced by treatment with Wnt1 protein. In C, quantitative results demonstrate that infarct size was significantly decreased by Wnt1 (24 µg/kg) treatment following reperfusion after cerebral ischemia. The total infarct size was expressed as a percentage of the contralateral hemisphere (*p < 0.05 vs. vehicle). In all cases, each data point represents the mean and SEM from six experiments.

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Expressing, Control, Injection, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Wnt1 neuroprotection translates into improved neurological function during oxidant stress and cerebral ischemia through AKT1 and mitochondrial apoptotic pathways

doi: 10.4161/oxim.3.2.9

Figure Lengend Snippet: Wnt1 signaling is required for reduction in cerebral infarction following transient cerebral MCAO and maintains neurological recovery. In A, B and C, focal cerebral ischemia was induced by insertion of a monofilament thread (4-0) into the internal carotid artery and blockade of the origin of MCA. Reperfusion was performed following 90 minutes ischemia by withdrawal of the thread. In A, Wnt1 protein (24 µg/kg), Wnt1Ab (60 µg/kg), or DKK-1 (30 µg/kg) was injected into the internal carotid artery through the external carotid artery at 30 min prior to the onset of MCAO and at the onset of reperfusion. Animals were euthanized 24 hours following ischemia and infarct size was determined by 2,3,5-triphenytetrazolium (TTC) staining. Representative images illustrate that infarct size (white in color) was significantly reduced by treatment with Wnt1 protein. In contrast, infarct size was markedly increased by treatment with Wnt1Ab or DKK-1 during MCAO. In B, quantitative results demonstrate that infarct size was significantly decreased by Wnt1 (24 µg/kg) treatment following reperfusion after MCAO. However, infarct size was substantially increased with Wnt1Ab (60 µg/kg) or DKK-1 (30 µg/kg) administration during MCAO. The total infarct size was expressed as a percentage of the contralateral hemisphere (*p < 0.05 vs. vehicle). In C, the neurological deficit score was assessed in animals 24 hours following MCAO and reperfusion of a 90 minute period. Wnt1 (24 µg/kg) significantly lowered the neurological deficit score when compared to vehicle only treated animals. In contrast, Wnt1Ab (60 µg/kg) or DKK-1 (30 µg/kg) significantly increased the neurological deficit score (*p < 0.05 vs. vehicle). In all cases, each data point represents the mean and SEM from six experiments.

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (Wnt1Ab) (R&D Systems, Minneapolis, MN), the phosphatidylinositol 3-kinase (PI-3 K) inhibitor wortmannin (Calbiochem, La Jolla, CA), the recombinant Wnt antagonist dickkopf related protein 1 (DKK-1, 0.5 μg/ml, R&D Systems, Minneapolis, MN), or D-3-deoxy-2-O-methyl- myo inositol 1-(R)-2-methoxy-3-(octadecyloxy) propyl hydrogen phosphate (SH-5) (Alexis, San Diego, CA) were continuous.

Techniques: Injection, Staining