Fig. 3. Wnt1 maintains inhibitory phosphorylation of p-FoxO3a and prevents nuclear translocation of FoxO3a in microglia during OGD.

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.