Figure 3 | Scientific Reports

Figure 3

From: Pharmacological mTOR targeting enhances the antineoplastic effects of selective PI3Kα inhibition in medulloblastoma

Figure 3

Dual PI3Kα and mTOR inhibition decreases the amount of nuclear GLI1 and exhibit potent antineoplastic effects in Ewing sarcoma cells. (A) DAOY medulloblastoma cells were treated with alpelisib (10 μM) and/or OSI-027 (5 μM) for 12 hours. After cell fractionation, nuclear and cytoplasmic extracts were subjected to immunoblotting using antibodies against GLI1, Lamin A/C and tubulin. Membranes were analysed using the ChemiDoc MP Imaging System. (B) TC71 Ewing Sarcoma cells were treated with alpelisib (0.15 μM) and/or OSI-027 (0.15 μM) for 24 hours and subjected to cell fractionation and immunoblotting using antibodies against GLI1, Lamin A/C and tubulin. Membranes were analysed using the ChemiDoc MP Imaging System. (C) RDES Ewing Sarcoma cells were treated with alpelisib (0.15 μM) and/or OSI-027 (0.15 μM) for 24 hours and subjected to cell fractionation and immunoblotting using antibodies against GLI1 and Lamin A/C. Membranes were stripped and reprobed with antibodies against HDAC1. Membranes were analysed using the ChemiDoc MP Imaging System. Uncropped blots for (A–C) are presented in the supplement. (D,E) TC71 (D) or RDES (E) Ewing Sarcoma cells were treated with alpelisib and/or OSI-027 at increasing concentrations for 5 days and cell viability was determined using the cell proliferation reagent, WST-1. Prism GraphPad 7.0 was used to determine IC50 values. Data represent means ± SEM of 3 independent experiments, each done in triplicate. (F,G) TC71 (F) or RDES (G) Ewing Sarcoma cells were seeded in soft agar in 96-well plates (5000 cells per well), treated with alpelisib (0.3 μM) and/or OSI-027 (0.3 μM). After 10 days, colony formation was quantified using the fluorescent CyQUANT GR Dye. Data represent means ± SEM of 3 independent experiments, each done in triplicate. Unpaired one-way ANOVA, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

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