Figure 4 | Scientific Reports

Figure 4

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

Figure 4

Inhibition of PI3Kα or PIK3CA knockdown reduces sphere formation and disrupts medulloblastoma stem cell frequencies when combined with pharmacologic mTOR inhibition. (A,B) DAOY (A) or D556 (B) cells were grown as spheres in CSC medium for 7 days. Spheres were dissociated and seeded at 500 cells/well into round-bottom 96-well plates in the presence of alpelisib (5 μM) and/or OSI-027 (1 μM). After 7 days, spheres were stained with acridine orange and imaged to determine cross-sectional area. Data represent means ± SEM of 3 independent experiments, each done in triplicate. Unpaired one-way ANOVA, **P ≤ 0.01, ****P ≤ 0.0001. Representative images are shown in the top panels. Scale bar, 1,000 μm. (C,D) In vitro ELDA after PIK3CA knockdown in combination with mTOR inhibition. DAOY (C) and D556 (D) cells were transfected with control siRNAs (siCtrl) or siRNAs targeting PIK3CA (siCA). After 2 days, cells were dissociated with trypsin and seeded in 3–5 technical replicates (n = 3) into round-bottom 96-well plates by forward- and side scatter, single-cell sorting at densities of 10, 30, 100, 300, 1,000 or 3,000 cells per well. Cells were treated with DMSO or OSI-027 (2 μM). After 7 days, neurospheres were stained with acridine orange and imaged using a Cytation 3 Cell Imaging multi-Mode Reader with a 4x objective. Neurospheres with a diameter of ≥100 μm were scored positive for ELDA analysis (http://bioinf.wehi.edu.au/software/elda/). (E,F) Stem cell frequencies of medulloblastoma stem-like cancer cells for DAOY (E) or D556 (F) were estimated as the ratio 1/x with the top and bottom 95% confidence intervals, where 1 = stem cell and x = all cells. (G,H) P values from χ2 analyses are shown for DAOY (G, left panel) and D556 (H, left panel). Whole cell lysates of DAOY (G, right panel) and D556 (H, right panel) were subjected to immunoblotting using antibodies against p110α to monitor knockdown of PIK3CA. Membranes were stripped and reprobed with antibodies against HSP90. Blots were analysed by autoradiography. Uncropped blots are presented in the supplement.

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