Metformin Suppresses Self-Renewal Ability and Tumorigenicity of Osteosarcoma Stem Cells via Reactive Oxygen Species-Mediated Apoptosis and Autophagy

Figure 3

Metformin activates the mitochondrial pathway to induce apoptosis. (a) Confocal laser scanning microscope of the mitochondrial morphology in K7M2 and MG63 OSCs by Mito Tracker. Nuclei were counterstained with DAPI. . . (b) Flow cytometry analysis of mitochondrial membrane potential in K7M2 and MG63 OSCs exposed to metformin. . (c) ATP concentrations in K7M2 and MG63 OSCs after metformin treatment for 48 h. . (d) Immunofluorescence analysis of ROS in K7M2 and MG63 OSCs using the DCFH-DA probe. Nuclei were counterstained with DAPI. . . (e) Measurement of intracellular ROS in K7M2 and MG63 OSCs labeled with DCFH-DA by flow cytometry. Cells were pretreated with 10 mM of the ROS scavenger NAC to eliminate ROS. . (f) Annexin V-FITC/PI staining of apoptosis in metformin-mediated K7M2 and MG63 OSCs by flow cytometry. . (g) The percentage of OSCs in early apoptosis and total apoptosis upon treatment of metformin. (h) Western blot analysis of mitochondrial apoptotic pathway-related proteins in metformin-treated K7M2 and MG63 OSCs. . (i) Densitometric analyses of mitochondrial apoptotic pathway-related proteins. was considered statistically significant.

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