Journal: Oxidative Medicine and Cellular Longevity
Article Title: Metformin Suppresses Self-Renewal Ability and Tumorigenicity of Osteosarcoma Stem Cells via Reactive Oxygen Species-Mediated Apoptosis and Autophagy
Figure Lengend Snippet: Characteristics of OSCs. (a) The representative images of SP cells from K7M2 and MG63 osteosarcoma cell lines. The median value of K7M2 and MG63 SP cells was 1.25% and 1.07%, respectively. n = 5. (b) The representative TEM images of autophagosomes in K7M2 and MG63 SP cells. The pentagrams stand for autophagosomes. Scale bars = 1 μ m. (c) Tumor spheres of K7M2 and MG63 osteosarcoma cells after culturing in the serum-free medium DMEM/F12-bFGF-EGF-B27 for 7 days. The parental K7M2 and MG63 cells cultured in DMEM/F12 supplemented with 1% FBS served as a control. Scale bars = 50 μ m. n = 5. (d) Western blot analysis of the pluripotent transcription factors Sox2, Oct4, and Nanog and the autophagy markers LC3, ATG5, and ATG7 in K7M2 and MG63 OSCs. Data are shown as mean ± SD, n = 3. (e) The mRNA expression levels of the pluripotency-associated genes SOX2 , OCT4 , and NANOG and the autophagy-related genes ATG5 and ATG7 . n = 3. (f) Immunofluorescence analysis of autophagy in K7M2 and MG63 SP cells. The colocalization (orange) staining of LC3 (green) with lysosome (red) indicates autophagy. Scale bars = 200 μ m. n = 3. (g) Osteogenic and chondrogenic differentiation of K7M2 and MG63 SP cells. Cells differentiated into osteoblasts and chondroblasts were detected by staining with Alizarin Red and Alcian Blue. Scale bars = 100 μ m. n = 3. (h) Flow cytometry-based assay for the pluripotent transcription factors Sox2 and Oct4 and the CSC surface markers CD44, CD105, CD133, and Stro-1 in K7M2 and MG63 SP cells. n = 3. ∗ P
Article Snippet: DAPI (BD5010) was obtained from Bioworld Technology, Inc. DMEM (12800082), DMEM/F12 (12400024), Fetal Bovine Serum (FBS, 10099-141), B27 Supplement (17504044), epidermal growth factor (EGF, PHG0311), and basic fibroblast growth factor (bFGF, 13256029) were purchased from Gibco (USA).
Techniques: Transmission Electron Microscopy, Cell Culture, Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry, Cytometry