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

Figure 2. Knockdown of p53 blocks the loss of stemness markers and FoxO proteins after combination treatment with γIR and PI-103.

Figure 2

(A) Assessment of shRNA knockdown efficiency by Western blot after 10-Gy γIR (shown are representative results from 3 experiments for each cell line; for statistical analysis, see Figure S4). (B) Control shRNA- or p53 shRNA-transduced GBM-SCs were incubated with PI-103 (1 μM) and irradiated 1 h later. Western blot analyses were performed after 3 or 7 days. Shown are representative results from 3 (GBM22) and 2 (GBM36) experiments, respectively. Quantification of changes in the expression of stem and progenitor markers and of FoxO proteins after combination treatment with 10 Gy + PI-103 compared to untreated cells; black columns: p53-proficient GBM-SCs; open columns: p53-knockdown cells. (C) Control or p53 knockdown GBM-SCs were treated with PI-103 (0.5 μM) for 1 h and irradiated with 10 Gy. Apoptosis was assessed at day 5 by flow-cytometric detection of annexin V staining, FSC/SSC analysis (n = 3 experiments), and Western blot for cleaved caspase 3 (1 of 2 experiments with similar results is shown). (D) Absolute cell numbers and sphere-forming capacity of control or p53 knockdown GBM-SCs after 3 days of incubation in CSC medium (n = 3 experiments). The bars represent 500 μm. Apoptosis and proliferation data in (C) and (D) represent means ± SD. KD, knockdown.