Figure 1.

Alpelisib and OSI-027 inhibit PI3K/mTOR signalling and exhibit antineoplastic effects in medulloblastoma cells. (A,B) DAOY (A) or D556 (B) cells were treated with alpelisib (10 μM) and/or OSI-027 (10 μM) for 90 minutes and subjected to immunoblotting with antibodies against phospho-4EBP1^T37/46 and phospho-S6K1^T389. Membranes were stripped and reprobed with antibodies for 4EBP1, S6K1 and GAPDH. Lysates from the same experiment were run in parallel and subjected to immunoblotting with antibodies against phospho-AKT^S473 followed by stripping and reprobing with antibodies for AKT. Blots were analysed by autoradiography. Uncropped blots are presented in the supplement. (C,D) DAOY (C) or D556 (D) cells in 96-well plates (2000 cells per well) 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 and CI values were calculated using Compusyn. Data represent means ± SEM of 3 independent experiments, each done in triplicate. (E,F) DAOY (E) or D556 (F) cells were seeded in soft agar in 96-well plates (1500 cells per well), treated with alpelisib (1 μM) and/or OSI-027 (1 μM). After 7 days, colony formation was quantified using the fluorescent CyQUANT GR Dye. Data represent means ± SEM of 5 independent experiments, each done in triplicate. Unpaired one-way ANOVA, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. (G,H) DAOY (G) or D556 (H) cells were treated with alpelisib (10 μM) and/or OSI-027 (2 μM). After 2 days, apoptosis was assessed by costaining cells with propidium iodide (PI) and annexin V-FITC followed by flow cytometry analysis. Annexin V positive cells were quantified to determine total apoptosis. Data represent means ± SEM of 4 (DAOY) and 5 (D556) independent experiments. Unpaired one-way ANOVA, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.