Oxidative Medicine and Cellular Longevity / 2016 / Article / Fig 3

Research Article

Plant Natural Product Formononetin Protects Rat Cardiomyocyte H9c2 Cells against Oxygen Glucose Deprivation and Reoxygenation via Inhibiting ROS Formation and Promoting GSK-3β Phosphorylation

Figure 3

Effects of formononetin on mitochondrial membrane integrity and phosphorylation of Akt and GSK-3β. (a) Fluorescence images and quantification of TMRM sequestration in mitochondria. H9c2 cells were treated with formononetin (5, 10, and 30 μM) under normal or OGD and reoxygenation condition and stained with TMRM. TMRM fluorescence intensity was determined by NIH image J software. Results are presented as means ± SD (). (OGD/R versus normal); , (drug versus OGD/R). Scale bar, 10 μm. (b) Fluorescence images and quantification of calcein sequestration in mitochondria. H9c2 cells were treated similarly as described in (a) and stained with calcein-AM. Calcein fluorescence intensity was determined by NIH image J software. Results are presented as means ± SD (). (OGD/R versus normal); (drug versus OGD/R). Scale bar, 10 μm. (c) Western blot analysis and quantification of phosphorylation of GSK-3β and Akt in response to formononetin. After the treatment with formononetin (5, 10, and 30 μM) under normal condition, H9c2 cells were lysed and subsequently analyzed for phosphorylation of GSK-3β (Ser9) and Akt by Western blotting using specific antibodies. Western blots were quantified by a densitometric method. Results are presented as means ± SD (). , (drug versus control). (d) Western blot analysis and quantification of phosphorylation of GSK-3β and Akt in response to formononetin under normal or OGD/R condition. After the treatment with formononetin (5, 10, and 30 μM) under normal or OGD/R condition, H9c2 cells were lysed and subsequently analyzed for phosphorylation of GSK-3β (Ser9) and Akt by Western blotting using specific antibodies. Western blots were quantified by a densitometric method. Results are presented as means ± SD (). ; (drugs versus control and drugs versus OGD/R).
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