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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. 6. WISP1 maintains mitochondrial membrane polarization while preventing cytochrome c release and the activation of caspase 3 through PI 3-K and Akt1 pathways during OGD.

Fig. 6

(A) Representative images and quantitative results from JC-1 staining reveal that OGD exposure produces a significant decrease in the red/green fluorescence intensity ratio of mitochondria within 3 hours when compared with untreated control cultures, demonstrating that mitochondrial membrane depolarization occurs 3 hours following OGD. WISP1 (10 ng/ml) 1 hour pretreatment significantly increased the red/green fluorescence intensity of mitochondria in neurons, demonstrating that mitochondrial membrane potential was restored. In contrast, inhibition of PI 3-K with wortmannin (0.5 μM) or LY294002 (10 μM) abrogated the ability of WISP1 to increase the mitochondrial membrane potential during OGD. The relative ratio of red/green fluorescent intensity of mitochondrial staining was measured in 3 independent experiments with analysis performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image) (*p<0.01 vs. OGD; †p<0.01 vs. WISP1/OGD). (B) Equal amounts of mitochondrial (mito) or cytosol (cyto) protein extracts (20 μg/lane) were immunoblotted with cytochrome c antibody demonstrating that WISP1 significantly prevented cytochrome c release from mitochondria 3 hours after a 3 hour period of OGD. The specific PI 3-K inhibitors wortmannin (0.5 μM) or LY294002 (10 μM) abrogated the ability of WISP1 to prevent cytochrome c release during OGD (*p<0.01 vs. OGD; †p <0.01 vs. WISP1/OGD). Quantification of the western band intensity was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nihimage). (C) Neuronal cell protein extracts (50 μg/lane) were immunoblotted with cleaved caspase 3 (active) antibody 3 hours after a 3 hour period of OGD. OGD significantly increased cleaved caspase 3 expression. In contrast, WISP1 (10 ng/ml) administered 1 hour prior to OGD markedly prevented the expression of cleaved caspase 3 during OGD. The specific PI 3-K inhibitors wortmannin (0.5 μM) or LY294002 (10 μM) blocked the ability of WISP1 to prevent caspase 3 activation (*p<0.01 vs. OGD; †p <0.01 vs. WISP1/OGD). Quantification of western band intensity from 3 experiments was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image).