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. Author manuscript; available in PMC: 2012 Feb 8.
Published in final edited form as: Curr Neurovasc Res. 2012 Feb 1;9(1):20–31. doi: 10.2174/156720212799297137

Fig. 3. WISP1 utilizes PI 3-K and Akt1 pathways to protect neurons against OGD.

Fig. 3

(A) Equal amounts of neuronal protein extracts (50 μg/lane) were immunoblotted at 1, 3 and 24 hours after administration of 5 ng/ml, 10 ng/ml or 20 ng/ml WISP1 with anti–phospho-Akt1 (p-Akt1, Ser473) antibody. WISP1 significantly enhanced p-Akt1 expression in a concentration dependent manner (*p<0.01 vs. Control). Quantitative analysis of the western blots from 3 experiments was performed using the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). (B) Equal amounts of neuronal protein extracts (50 μg/lane) were immunoblotted with p-Akt1 antibody at 1, 3 and 24 hours following a 3 hour period of OGD. The expression of p-Akt1 was increased at 1 and 3 hours following OGD exposure and was further increased by WISP1 (10 ng/ml) administration 1 hour prior to OGD (*p<0.01 vs. Control; †p <0.01 vs. OGD). (C) Application of the specific PI 3-K inhibitors wortmannin (0.5 μM) or LY294002 (10 μM) 1 hour prior to a 3 hour period of OGD abrogated WISP1 induced expression of p-Akt1 3 hours following a 3 hour period of OGD (*P<0.01 vs. Control; †P <0.01 vs. WISP1/OGD). (D) Representative images demonstrate that OGD led to a significant increase in trypan blue staining and DNA fragmentation in neuronal cells 24 hours after OGD compared to untreated control cultures. WISP1 (10 ng/ml) application 1 hour prior to OGD significantly decreased trypan blue and TUNEL staining. Yet, combined treatment with specific PI 3-K inhibitors wortmannin (0.5 μM) or LY294002 (LY, 10 μM) abrogated the ability of WISP1 to reduce trypan blue staining and DNA fragmentation. Quantitative results illustrate that WISP1 (10 ng/ml) application significantly decreased percent trypan blue uptake and DNA fragmentation 24 hours after OGD when compared to OGD. Combined treatment with specific PI 3-K inhibitors wortmannin or LY294002 significantly reduced the efficacy of WISP1, resulting in an increase in percent trypan blue uptake and DNA fragmentation (*p <0.01 vs. untreated control; †P <0.01 vs. OGD). Each data point represents the mean and SEM from 6 experiments.