mouse antibody against wnt1  (Proteintech)

Journal: Metabolites

Article Title: Exercise Promotes the Osteoinduction of HA/β-TCP Biomaterials via the Wnt Signaling Pathway

doi: 10.3390/metabo10030090

Figure Lengend Snippet: ( A ) Western blot showing the expression of wingless/integrated 1 (Wnt1), Wnt3a, Wnt5a, β-catenin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in five groups ( n = 3). G1: negative control group; G2: running group; G3: weight-bearing group; G4: positive control group; Neonatal: the neonatal bone, control. ( B ) The corresponding gray-scale value of Wnt1, Wnt3a, Wnt5a and β-catenin in the five groups ( n = 3); comparison among G1 and other three groups (G3, G4, and Neonatal), * P < 0.05.

Article Snippet: After transferring the proteins onto a nitrocellulose membrane, the membrane was blocked with a 5% defatted milk solution and probed with mouse antibody against Wnt1 (1:1000, Proteintech, Rosemont, IL, USA), Wnt3a (1:1000, AFFINITY, Cincinnati, OH, USA), Wnt5a (1:600, Proteintech), β-catenin (1:1000, AFFINITY), runt-related transcription factor-2 (RUNX-2, 1:600, AFFINITY), osteopontin (OPN, 1:2000, Proteintech), osteocalcin (OCN, 1:1000, AFFINITY), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, Santa Cruz, Santa Cruz, CA, USA), and then probed with a secondary antibody using ALP conjugated anti-mouse IgG (1:5000, Santa Cruz, USA).

Techniques: Western Blot, Expressing, Negative Control, Positive Control

Journal:

Article Title: WNT1, FoxO3a, AND NF-?B OVERSEE MICROGLIAL INTEGRITY AND ACTIVATION DURING OXIDANT STRESS

doi: 10.1016/j.cellsig.2010.04.009

Figure Lengend Snippet: (A and B) Microglial 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, but then progressively and significantly is reduced at 6 and 24 hours following OGD exposure (*P<0.01 vs. control). (C) Microglia were exposed to progressive durations of OGD at 4, 6, 8, and 12 hours and microglial survival was determined 24 hours later by trypan blue dye exclusion assay. Microglial survival was significantly decreased to 58 ± 4% (4 hours), 38 ± 4% (6 hours), 32 ± 4% (8 hours), and 27 ± 3% (12 hours) following OGD exposure when compared with untreated control cultures (92 ± 3%, *P <0.01 vs. Control). Each data point represents the mean and SEM from 6 experiments. (D and E) Wnt1 (100 ng/ml) was administered 1, 12, 24, or 48 hours prior to a 6 hour period of OGD. 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 application at each time period. Quantification of data demonstrates that OGD significantly decreased percent cell survival when compared to the control cells. Wnt1 significantly increased cell survival at each time period, but application of Wnt1 closest to the point of injury at the 1 hour period yielded the greatest degree of cytoprotection for microglia (*P<0.01 vs. control; †P<0.01 vs. OGD). Each data point represents the mean and SEM from 6 experiments. Control = untreated microglia.

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (R&D Systems, Minneapolis, MN) were continuous.

Techniques: Expressing, Exclusion Assay, Staining

Journal:

Article Title: WNT1, FoxO3a, AND NF-?B OVERSEE MICROGLIAL INTEGRITY AND ACTIVATION DURING OXIDANT STRESS

doi: 10.1016/j.cellsig.2010.04.009

Figure Lengend Snippet: (A and B) Microglial cells were exposed to a 6 hour period of OGD and microglial survival was determined 24 hours later by trypan blue assay. Representative images illustrate increased trypan blue staining during OGD and during blockade of Wnt1 with Wnt1Ab (1 μg/ml) and combined Wnt1 (100 ng/ml) administration. Wnt1 (100 ng/ml) administration alone significantly increased microglial survival during OGD. In addition, Wnt1Ab (1 μg/ml) alone markedly decreased microglial survival during OGD to a greater degree than OGD alone, suggesting that an endogenous level of Wnt1 in microglia is protective against cell injury. In all cases control = untreated microglia (*P<0.01 vs. OGD; †P <0.01 vs. Wnt1/OGD). Each data point represents the mean and SEM from 6 experiments. (C and D) Representative images illustrate that Wnt1 (100 ng/ml) administration during OGD significantly blocks microglial genomic DNA degradation assessed by TUNEL and membrane PS externalization assessed by annexin V phycoerythrin (green fluorescence). In contrast, blockade of Wnt1 with Wnt1Ab (1 μg/ml) resulted in increased DNA fragmentation and membrane PS exposure and higher apoptotic injury than OGD alone in the presence of Wnt1Ab (1 μg/ml) only, suggesting that an endogenous level of Wnt1 also provides protection against apoptotic early and late programs. Quantification of data illustrates that DNA fragmentation and membrane PS externalization were significantly increased following OGD for a 6 hour period when compared to untreated microglial control cultures, but Wnt1 (100 ng/ml) prevents DNA fragmentation and membrane PS exposure during OGD (*P<0.01 vs. OGD; †P <0.01 vs. Wnt1/OGD). Inhibition of Wnt1 with Wnt1Ab (1 μg/ml) significantly worsens apoptotic injury. Each data point represents the mean and SEM from 6 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 (R&D Systems, Minneapolis, MN) were continuous.

Techniques: Staining, TUNEL Assay, Membrane, Fluorescence, Inhibition

Journal:

Article Title: WNT1, FoxO3a, AND NF-?B OVERSEE MICROGLIAL INTEGRITY AND ACTIVATION DURING OXIDANT STRESS

doi: 10.1016/j.cellsig.2010.04.009

Figure Lengend Snippet: In A and B, microglial protein extracts (50 μg/lane) were immunoblotted with anti-phosphorylated-FoxO3a (p-FoxO3a, Ser253) or anti-total FoxO3a at 1 and 6 hours following OGD exposure. Quantification of western band intensity was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image). During OGD, phosphorylated (inactive) FoxO3a (p-FoxO3a) expression is significantly decreased at 1 hour and 6 hours following OGD, but total FoxO3a expression not affected illustrating that FoxO3a protein is intact but post-translational phosphorylation has been changed (*P<0.01 vs. control). Wnt1 (100 ng/ml) administration increases phosphorylation of FoxO3a at 1 hour when compared to this time period with OGD only and significantly increases inhibitory phosphorylation of FoxO3a at 6 hours following OGD exposure (*P<0.01 vs. untreated microglia = Control; †P <0.01 vs. OGD at 1 hour). In C and D, equal amounts of cytoplasmic (cytoplasm) or nuclear (nucleus) protein extracts (50 μg/lane) were immunoblotted with anti-FoxO3a at 6 hours following administration of OGD. At 6 hours following OGD alone, FoxO3a translocates from the cytoplasm to the nucleus. In contrast, Wnt1 (100 ng/ml) prevents trafficking of FoxO3a from the cytoplasm to the cell nucleus in microglia (*P<0.01 vs. untreated microglia = Control; †P <0.01 vs. OGD). In E and F, microglia were imaged 6 hours following OGD with immunofluorescent staining for FoxO3a (Texas-red streptavidin). Nuclei of microglia were counterstained with DAPI. In merged images, untreated control microglia have visible nuclei (dark blue in color, white arrows) that illustrate absence of FoxO3a in the nucleus. Merged images after OGD demonstrate microglia with red cytoplasm (green arrows) and no visible nucleus with DAPI illustrating translocation of FoxO3a to the nucleus. Wnt1 (100 ng/ml) application during OGD maintains FoxO3a in the cytoplasm of microglia (*P<0.01 vs. untreated microglia = Control; †P <0.01 vs. OGD). In D and F, quantification of the intensity of FoxO3a nuclear staining or FoxO3a western expression was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image). Each data point represents the mean and SEM from 6 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 (R&D Systems, Minneapolis, MN) were continuous.

Techniques: Western Blot, Expressing, Staining, Translocation Assay

Journal:

Article Title: WNT1, FoxO3a, AND NF-?B OVERSEE MICROGLIAL INTEGRITY AND ACTIVATION DURING OXIDANT STRESS

doi: 10.1016/j.cellsig.2010.04.009

Figure Lengend Snippet: In A and B, microglial protein extracts (50 μ/lane) were immunoblotted with anti-total FoxO3a at 6 hours following OGD. Gene knockdown of FoxO3a was performed with transfection of FoxO3a siRNA (siRNA). FoxO3a siRNA significantly reduced expression of total FoxO3a following a 6 hour period of OGD or during Wnt1 (100 ng/ml) application with OGD, but non-specific scrambled siRNA did not alter total FoxO3a expression (*P<0.01 vs. OGD). Quantification of the western band intensity was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image). In C and D, gene knockdown of FoxO3a with FoxO3a siRNA (siRNA) significantly increased microglial survival and decreased microglial membrane injury assessed by trypan blue staining 24 hours after OGD in representative figures and quantitative analysis. In addition, significantly increased microglial cell survival is present during Wnt1 (100 ng/ml) administration with gene knockdown of FoxO3a similar to gene knockdown of FoxO3a alone during OGD. FoxO3a siRNA alone was not toxic and non-specific scrambled siRNA did not protect cells during OGD (*P<0.01 vs. untreated microglia = Control; †P <0.01 vs. OGD). Each data point represents the mean and SEM from 6 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 (R&D Systems, Minneapolis, MN) were continuous.

Techniques: Transfection, Expressing, Western Blot, Membrane, Staining

Journal:

Article Title: WNT1, FoxO3a, AND NF-?B OVERSEE MICROGLIAL INTEGRITY AND ACTIVATION DURING OXIDANT STRESS

doi: 10.1016/j.cellsig.2010.04.009

Figure Lengend Snippet: In A, representative images illustrate gene knockdown of FoxO3a with FoxO3a siRNA (siRNA) significantly blocks microglial genomic DNA degradation assessed by TUNEL and membrane PS externalization assessed by annexin V phycoerythrin (green fluorescence) 24 hours after OGD. Non-specific scrambled siRNA did not alter DNA fragmentation or membrane PS exposure. In addition, combined administration of Wnt1 (100 ng/ml) during gene knockdown of FoxO3a also resulted in a similar degree of protection against DNA fragmentation and membrane PS exposure in microglia when compared to gene knockdown of FoxO3a alone, suggesting that Wnt1 requires FoxO3a inhibition for the prevention of apoptotic programs. In B, quantification of data illustrates that DNA fragmentation and membrane PS externalization were significantly increased following OGD when compared to untreated microglial control cultures, but transfection of FoxO3a siRNA alone or in combination with Wnt1 (100 ng/ml) prevents DNA fragmentation and membrane PS exposure during OGD (*P<0.01 vs. untreated microglia = Control; †P <0.01 vs. OGD). FoxO3a siRNA alone was not toxic and non-specific scrambled siRNA did not protect cells during OGD. Each data point represents the mean and SEM from 6 experiments. In C, representative images illustrate that PCNA and BrdU expression is significantly and rapidly increased in microglia at 6 hours after OGD. Wnt1 (100 ng/ml) alone or in combination with gene knockdown of FoxO3a with FoxO3a siRNA (siRNA) significantly decreases the expression of PCNA and the uptake of BrdU at 6 hours after OGD. In D, quantification of data demonstrates that PCNA and BrdU were significantly increased following OGD (*p<0.01 vs. untreated microglia = control). In addition, Wnt1 (100 ng/ml) alone or in combination with gene knockdown of FoxO3a with FoxO3a siRNA (siRNA) markedly reduces the expression of PCNA and the uptake of BrdU at 6 hours after OGD (*P<0.01 vs. untreated microglia = control; †P <0.01 vs. OGD). FoxO3a siRNA alone was not toxic and non-specific scrambled siRNA did not alter PCNA expression or BrdU uptake during OGD (†P <0.01 vs. OGD). In all cases, control = untreated cells. Each data point represents the mean and SEM from 6 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 (R&D Systems, Minneapolis, MN) were continuous.

Techniques: TUNEL Assay, Membrane, Fluorescence, Inhibition, Transfection, Expressing

Journal:

Article Title: WNT1, FoxO3a, AND NF-?B OVERSEE MICROGLIAL INTEGRITY AND ACTIVATION DURING OXIDANT STRESS

doi: 10.1016/j.cellsig.2010.04.009

Figure Lengend Snippet: 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 6 hours when compared with untreated control microglial cells, 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 microglia, illustrating that mitochondrial membrane potential was restored. Furthermore, transfection of FoxO3a siRNA alone or in combination with Wnt1 (100 ng/ml) also maintained mitochondrial membrane potential similar to Wnt1 administration alone. Transfection with non-specific scrambled siRNA did not prevent mitochondrial membrane depolarization during OGD. In B, the relative ratio of red/green fluorescent intensity of mitochondrial staining in untreated control microglia, in microglia exposed to a 6 hour period of OGD, during Wnt1 administration or during Wnt1 administration with gene knockdown of FoxO3a, or during FoxO3a gene knockdown alone was measured in 6 independent experiments with analysis performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image) (untreated microglia = Control vs. OGD, *P<0.01; Wnt1 or Wnt1 plus siRNA FoxO3a vs. OGD, †P<0.01). In C, equal amounts of mitochondrial (mito) or cytosol (cyto) protein extracts (50 μg/lane) were immunoblotted demonstrating that Wnt1 (100 ng/ml) administration alone, in combination with gene knockdown of FoxO3a, or gene knockdown of FoxO3a alone significantly prevented cytochrome c release from mitochondria 6 hours after OGD. Non-specific scrambled siRNA did not prevent cytochrome c release during OGD. In D, quantification of the western band intensity was performed using the public domain NIH image program (http://rsb.info.nih.gov/nih-image) and demonstrates that significant release of cytochrome c occurs 6 hours following OGD, but Wnt1 (100 ng/ml) administration alone or during Wnt1 administration with gene knockdown of FoxO3a prevents cytochrome c release from microglial mitochondria. Non-specific scrambled siRNA was ineffective during OGD to prevent cytochrome c release (untreated microglia = Control vs. OGD, *P<0.01; Wnt1 or Wnt1 plus siRNA FoxO3a vs. OGD, †P<0.01). Each data point represents the mean and SEM from 6 experiments. In E and F, primary microglial protein extracts (50 μ/lane) were immunoblotted with anti-phosphorylated-Bad (p-Bad, Ser136) at 6 hours following OGD. Phosphorylated Bad (p-Bad) expression is promoted by Wnt1 (100 ng/ml) administration alone, during Wnt1 administration with gene knockdown of FoxO3a, or during gene knockdown of FoxO3a alone, but is significantly diminished during OGD alone. Non-specific scrambled siRNA during OGD did not change Bad phosphorylation and was similar to Bad phosphorylation during OGD alone (untreated microglia = Control vs. OGD, *P<0.01; Wnt1 or Wnt1 plus siRNA FoxO3a vs. 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 (R&D Systems, Minneapolis, MN) were continuous.

Techniques: Fluorescence, Membrane, Transfection, Staining, Western Blot, Expressing

Journal:

Article Title: WNT1, FoxO3a, AND NF-?B OVERSEE MICROGLIAL INTEGRITY AND ACTIVATION DURING OXIDANT STRESS

doi: 10.1016/j.cellsig.2010.04.009

Figure Lengend Snippet: In A, microglia were exposed to OGD and caspase 3 and caspase 1 activation were determined 6 hours after OGD period through immunocytochemistry with antibodies against cleaved active caspase 3 (17 kDa) and cleaved active caspase 1 (20 kDa). Representative images illustrate active caspase 3 staining or caspase 1 staining (red) in cells following OGD, but cellular red staining is almost absent during Wnt1 (100 ng/ml) administration alone or during Wnt1 administration with gene knockdown of FoxO3a. Non-specific scrambled siRNA did not eliminate caspase 3 or caspase 1 activity during OGD. In B, quantification of caspase 3 and caspase 1 immunocytochemistry was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image) and demonstrates that OGD significantly increased the expression of cleaved (active) caspase 3 or caspase 1 when compared to untreated control cells. Expression of cleaved (active) caspase 3 or caspase 1 was significantly limited during Wnt1 (100 ng/ml) administration alone or during Wnt1 administration with gene knockdown of FoxO3a. (*P <0.01 vs. untreated microglia = Control; †P<0.01 vs. OGD). In C, microglial protein extracts (50 μg/lane) were immunoblotted with anti-cleaved caspase 3 product (active caspase 3, 17 kDa) and with anti-cleaved caspase 1 product (active caspase 1, 20 kDa) at 6 hours following OGD. OGD markedly increased cleaved caspase 3 and caspase 1 expression, but Wnt1 (100 ng/ml) administration alone or during Wnt1 administration with gene knockdown of FoxO3a significantly blocked cleaved caspase 3 and caspase 1 expression 6 hours after OGD. Non-specific scrambled siRNA did not eliminate caspase 3 or caspase 1 activities during OGD. In D, quantification of western band intensity was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image) and demonstrates that Wnt1 (100 ng/ml) application alone or Wnt1 administration in combination with gene knockdown of FoxO3a prevents cleaved caspase 3 and caspase 1 expression 24 hours after OGD (*P <0.01 vs. untreated microglia = Control; †P<0.01 vs. OGD). Each data point represents the mean and SEM from 6 experiments. In E, microglial protein extracts (50 μg/lane) were immunoblotted with anti-cleaved caspase 3 product (active caspase 3, 17 kDa) and with anti-cleaved caspase 1 product (active caspase 1, 20 kDa) at 24 hours following OGD. OGD markedly increased cleaved caspase 3 and caspase 1 expression, but gene knockdown of FoxO3a with FoxO3a siRNA (siRNA) significantly blocks cleaved (active) caspase 3 and caspase 1 activities 24 hours after OGD. Non-specific scrambled siRNA did not eliminate caspase 3 or caspase 1 activities during OGD. In F, quantification of western band intensity was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image) and demonstrates that gene knockdown of FoxO3a prevents cleaved caspase 3 and caspase 1 expression 6 hours after OGD. In addition, non-specific scrambled siRNA did not change caspase 1 and caspase 3 activities when compared to OGD alone (*P <0.01 vs. untreated microglia = Control; †P<0.01 vs. OGD). Each data point represents the mean and SEM from 6 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 (R&D Systems, Minneapolis, MN) were continuous.

Techniques: Activation Assay, Immunocytochemistry, Staining, Activity Assay, Expressing, Western Blot

Journal:

Article Title: WNT1, FoxO3a, AND NF-?B OVERSEE MICROGLIAL INTEGRITY AND ACTIVATION DURING OXIDANT STRESS

doi: 10.1016/j.cellsig.2010.04.009

Figure Lengend Snippet: In A and B, microglia were imaged 6 hours following OGD with immunofluorescent staining for NF-κB p65 (Texas-red streptavidin). Nuclei of microglia were counterstained with DAPI. In merged images, untreated control microglia do not have visible nuclei (red in color, white arrows) that illustrate the presence of NF-κB p65 in the nucleus. Merged images after OGD demonstrate microglia with visible nuclei (light pink in color, white arrows) and red cytoplasm (green arrows) demonstrating that NF-κB p65 is retained in the cytoplasm. Wnt1 (100 ng/ml) administration during OGD or during Wnt1 administration with gene knockdown of FoxO3a during OGD fosters the translocation of NF-κB p65 to the cell nucleus. Non-specific scrambled siRNA does not alter NF-κB p65 subcellular trafficking during OGD (*P<0.01 vs. untreated microglia = Control; †P <0.01 vs. OGD). In C and D, equal amounts of cytoplasmic (cytoplasm) or nuclear (nucleus) protein extracts (50 μg/lane) were immunoblotted with anti- NF-κB p65 at 6 hours following administration of OGD. NF-κB p65 is maintained in the cytoplasm of microglia during OGD. In contrast, Wnt1 (100 ng/ml) administration with OGD or during Wnt1 administration with gene knockdown of FoxO3a with OGD allows trafficking of NF-κB p65 from the cytoplasm to the cell nucleus in microglia (*P<0.01 vs. untreated microglia = Control; †P <0.01 vs. OGD). In E and F, equal amounts of cytoplasmic (cytoplasm) or nuclear (nucleus) protein extracts (50 μg/lane) were immunoblotted with anti-NF-κB p65 at 6 hours following administration of OGD. As previously shown, NF-κB p65 is maintained in the cytoplasm of microglia during OGD. Yet, gene knockdown of FoxO3a during OGD fosters trafficking of NF-κB p65 from the cytoplasm to the cell nucleus in microglia. Non-specific scrambled siRNA did not alter NF-κB p65 translocation during OGD alone (*P<0.01 vs. untreated microglia = Control; †P <0.01 vs. OGD). In B, D, and F, quantification of the intensity of FoxO3a nuclear staining or NF-κB p65 expression on western was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image). Each data point represents the mean and SEM from 6 experiments. In G and H, microglial protein extracts (50 μ/lane) were immunoblotted with anti- NF-κB p65 at 6 hours following OGD. Gene knockdown of NF-κB p65 was performed with transfection of NF-κB p65 siRNA (siRNA). NF-κB p65 siRNA significantly reduced expression of NF-κB p65 following a 6 hour period of OGD or during Wnt1 (100 ng/ml) application with OGD, but non-specific scrambled siRNA did not alter NF-κB p65 expression (*P<0.01 vs. OGD). Quantification of the western band intensity was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image). In I and J, representative images and quantitative analysis show that gene knockdown of NF-κB p65 with NF-κB p65 siRNA (siRNA) significantly decreased microglial survival and increased microglial membrane injury assessed by trypan blue staining 24 hours after OGD. Loss of microglial cell survival also is present during Wnt1 (100 ng/ml) administration with gene knockdown of NF-κB p65 during OGD. In addition, gene knockdown of NF-κB during OGD alone resulted in increased cell injury when compared to microglial injury during OGD alone, illustrating that endogenous levels of NF-κB are cytoprotective during oxidative stress. Transfection with scrambled siRNA did not alter microglial injury during OGD or during OGD with Wnt1 (100 ng/ml) administration (*P<0.01 vs. OGD).

Article Snippet: For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) or mouse monoclonal anti body against Wnt1 (R&D Systems, Minneapolis, MN) were continuous.

Techniques: Staining, Translocation Assay, Expressing, Western Blot, Transfection, Membrane

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

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, 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

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

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, 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, 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