Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2019 Sep 6;9:12822. doi: 10.1038/s41598-019-49299-3

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

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.