Proteomic Analysis of Hypoxia-Induced Senescence of Human Bone Marrow Mesenchymal Stem Cells

Figure 7

The activation of the PI3K antagonizes the effects of CCND1 on reactive oxygen species (ROS) production and apoptosis in hypoxia-induced senescent hBMSCs. (a) Hypoxia promotes ROS generation, which was reduced by pretreatment with 740 Y-P. Fluorescein isothiocyanate- (FITC-) labeled 2,7-dichlorodihydrofluorescein acetoacetic acid (DCFH-DA) was used to detect intracellular ROS by flow cytometry, and mean fluorescence intensity (MFI) in cells was calculated. (b) The percentage of apoptotic cells was measured by Annexin V/7-AAD staining. Cells apoptosis was significantly decreased by 740 Y-P treatment compared to the hypoxia-induced group. (c) Total PI3K (t-PI3K), phospho-PI3K (p-PI3K), total CCND1 (t-CCND1), and phospho-CCND1 (p-CCND1) protein expressions were analyzed by western blotting and quantified by densitometry. The p-PI3K/t-PI3K ratio in hypoxia-induced senescent cells was significantly higher than that of the control group; however, the p-CCND1/t-CCND1 ratio was decreased after hypoxia induction. . vs. the 0 h control group. ^## vs. the 12-hour hypoxia-induced group. Western blot bands of p-PI3K (110 kDa), t-PI3K (110 kDa), p-CCND1 (36 kDa), t-CCND1 (36 kDa), and GAPDH (37 kDa). Experiments were performed in three hBMSC cultures from three different donors.

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