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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: Anti-EMT properties of CoQ0 attributed to PI3K/AKT/NFKB/MMP-9 signaling pathway through ROS-mediated apoptosis

Fig. 3

CoQ0 inhibits metastasis through the downregulation of PI3K/AKT/NFκB signaling pathways in MDA-MB-231 cells. a Cells were grown in chamber slides and exposed to CoQ0 (0.5–2 μM) for 2 h, fixed and permeabilized. Cells were incubated with anti-p65 antibody followed by FITC-labeled secondary antibody. The subcellular localization of p65 was visualized using a confocal microscope of 40 × magnification. b NFκB activity was evaluated via luciferase reporter gene assay. Cells were transfected with the luciferase reporters and co-transfected with either NFκB or empty vector. Then, they were treated with CoQ0 (0.5–2 μM) for 4 h, and luciferase activity was determined and normalized with β-gal activity, shown as relative luciferase activity. c CoQ0 induces nuclear p65 and inhibits cytosolic I-κB and p-IKK degradation. Cells were treated with CoQ0 (0.5–2 μM for 2 h). Cytoplasmic and nuclear extracts were prepared and Western blot analyses were performed. β-actin or histone H3 was used as an internal control for cytoplasmic and nuclear extracts, respectively. d CoQ0 inhibits MMP-9 expression through the downregulation of NFκB signaling pathways. The nuclear p65 (for 4 h) and MMP-9 expression for 24 h was inhibited following the treatment with CoQ0 (1 μM) in the presence or absence of NFκB inhibitor celastrol (0.3 μM), as shown by Western blot. For internal control β-actin was used. e Phosphorylated PI3K (p-PI3K) and AKT (p-AKT) levels were evaluated by immunoblot analysis. Cells were treated with CoQ0 (0.5–2 μM) for 24 h. β-actin, total PI3K and AKT levels were considered as an internal control. f CoQ0 inhibits NFκB/MMP-9 expression through the downregulation of PI3K/AKT signaling pathways. The nuclear p65 (for 4 h) and MMP-9 expression (for 24 h) was inhibited following treatment with CoQ0 (1 μM) in the presence of absence of PI3K/AKT inhibitor LY294002 (25 μM), as shown by Western blot. β-actin was used as an internal control. The results are presented as the mean ± SD of three independent assays. **p < 0.05, ***p < 0.001 significant compared to control cells

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