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

Figure 6

Autophagy regulates the homeostasis of pluripotency in OSCs. (a) TEM images of autophagosomes in metformin-treated K7M2 and MG63 OSCs. The pentagrams stand for autophagosomes. . (b) Immunofluorescence analysis of autophagy in K7M2 and MG63 OSCs. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. . . (c) Flow cytometry analysis of the effect of autophagy on ALDH1 activity. The upper panel shows that OSCs were incubated with DEAB, a specific ALDH inhibitor. The lower panel shows ALDH1-positive OSCs. . (d) The percentage of ALDH1-positive OSCs. (e) Clone formation of K7M2 and MG63 OSCs. . . (f) SEM images of sphere forming of K7M2 and MG63 OSCs. . . (g) Western blot analysis of the autophagy markers ATG5 and ATG7 and the pluripotency-associated proteins Sox2 and Oct4 in K7M2 and MG63 OSCs. . (h) Densitometric analyses of autophagy markers and pluripotency-associated proteins. Each experiment was conducted at least three occasions. was considered statistically significant.

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