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. 2016 Jul 19;7(34):54883–54896. doi: 10.18632/oncotarget.10702

Figure 6. FoxO3 overexpression inhibits the downregulation of stemness markers after combination treatment with γIR and PI-103 in p53-proficient GBM-SCs.

Figure 6

(A and E, left) Western blot analysis of the expression of FoxO proteins, stem cell markers, and GFAP in GBM-SCs transduced with control or FoxO3-encoding lentiviral particles (n = 3 experiments; for statistical analysis, see Figure S4). (A and E, right) Absolute cell numbers after 2 or 4 days of incubation in CSC medium. (B and F, left) Confirmation of the transduction efficiency by RFP expression analysis. The bar represents 100 μm. (B and F, right) Sphere-forming capacity after 3 days of incubation in CSC medium. The bar represents 500 μm. (C and G) Assessment of cleaved caspase 3 (1 of 2 experiments is shown, each with similar results) and of apoptosis by annexin V staining. Cells were treated with 0.5 μM PI-103 for 1 h and then irradiated with 10 Gy. The analyses were performed after 2 (GBM22) or 6 days (GBM4). (D) Western blot analyses of control or FoxO3-overexpressing GBM22-SCs showing that FoxO3 overexpression inhibits the downregulation of stemness markers in p53-proficient GBM-SCs (1 of 2 experiments is shown, each with similar results). The cells were treated as indicated in (C) and (G) and analyzed after 2 days. Data for cell and sphere numbers and apoptosis in (A)–(C) and (E)–(G) represent means ± SD from 3 independent experiments. OE, overexpressing.