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. 2017 Apr 18;2017:7371615. doi: 10.1155/2017/7371615

Figure 3.

Figure 3

Akt-cyclin D1 pathway activation is responsible for the pro-proliferative effect of insulin in UCM-MSC. (a) Specific activation of Akt in UCM-MSC following insulin treatment. Western blot analysis of phosphorylated Akt and ERK (p-Akt/ERK) in UCM-MSC exposed to 0, 5, and 10 μM insulin for 72 h under serum-deprived conditions. Left, representative blots showing the phosphorylated Akt and ERK protein levels following exposure to the indicated concentrations of insulin (GAPDH served as an internal control for protein loading); right, bar plot showing p-ERK/GAPDH and p-Akt/GAPDH ratios, as evaluated by densitometric analysis of Western blots and normalized to the untreated control. Data are shown as mean ± SD, and error bars indicate SD (n = 3). ∗p < 0.05 versus nontreated control cells by one-way ANOVA with Fisher's test (ns, not significant between indicated groups). (b) Decreases in insulin-induced cyclin D1 and phosphorylated Akt expression in UCM-MSC after Akt inactivation. UCM-MSC cultured in SFM were either left untreated or treated with an Akt inhibitor (LY294002, 5 μM) in the presence and absence of 10 μM insulin for 72 h and then subjected to Western blot analysis with anti-cyclin D1, anti-p-Akt, and anti-GAPDH (loading control) antibodies. Left, representative blots showing the phosphorylated Akt and cyclin D1 protein levels following the indicated treatments; right, bar plots showing p-Akt/GAPDH and cyclin D1/GAPDH ratios as assessed by densitometric analysis of Western blots and normalized to the untreated control. (c) Akt inhibition attenuates the pro-proliferative effect of insulin. UCM-MSC cultured in SFM were subjected to different treatments as described in (b), and cell proliferation was measured by CCK-8 assays. Values are adjusted relative to the proliferation of the nontreatment control, which was set to 100%. For bar graphs in (b) and (c), data are shown as mean ± SD, and error bars indicate SD (n = 3). Asterisks denote significant differences between indicated groups by Student's t-test (p < 0.05).