Figure 3
Effects of dual PI3Kα and mTOR inhibition in GSCs. (A–D) 83Mes (A), JK16 (B), 157PN (C), and AC17PN (D) GSC lines were seeded at 500 cells per well into round-bottom 96-well plates in the presence of increasing concentrations of alpelisib as indicated. After 7 days, spheres were stained with acridine orange and imaged on a Cytation3 instrument using Gen5 v 2.09 software to determine cross-sectional area. Prism GraphPad 8 was used to determine IC50 values. Data represent means ± SEM of 4 (83Mes, JK16), or 3 (157PN, AC17PN) independent experiments, each done in triplicate. Representative images are shown in the top panels. (E–H) 83Mes (E), JK16 (F), 157PN (G), and AC17PN (H) GSC lines were seeded at 500 cells per 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 4 (83Mes, JK16), 3 (157PN), and 5 (AC17PN) independent experiments, each done in triplicate. Representative images are shown in the top panels. Unpaired one-way ANOVA, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. (I-P) GSCs were subjected to in vitro limiting dilution assays (n = 3, each done in duplicate) plating decreasing number of cells (500; 100; 50; 10; 5; 1 cells per well) in the presence of alpelisib (5 μM) and/or OSI-027 (1 μM). ELDA for 83Mes (I,M), JK16 (J,N), 157PN (K,O), and AC17PN (L,P) was done using the ELDA software (http://bioinf.wehi.edu.au/software/elda/). (M-P, upper panels) Stem cell frequencies of GSCs were estimated as the ratio 1/x with the upper and lower 95% confidence intervals, where 1 = stem cell and x = all cells. (M-P, lower panels) P-values from chi-square analysis of group comparisons.
