Figure 6

Effects of CAR on the frequency of U87MG sphere-forming cells and self-renewal capacity. (A–C) U87MG-derived CSCs were grown in 0.36% agar in NSC medium in the presence of DMSO (0,5%, CTRL) or CAR (1–20 µM) for 14 days. At the end of the incubation, representative microscopic images (A) were captured at ×4 magnification before and after crystal violet staining. The number (B) and mean area (C) of the colonies were scored using the ImageJ program. Only colonies with diameters greater than 60 µm were counted. The data are presented as the mean values from three independent experiments. For each experimental condition, five pictures were analyzed. (D) A single U87MG-derived CSC was cultured in a 96-well plate and maintained in NSC medium for 3 weeks in the absence (CTRL) or presence of different concentrations of CAR (100 nM–10 µM). Images were captured at different time points. (E) The U87MG-derived CSCs were treated with DMSO (CTRL) or CAR (20) in NSC medium for 7 days. At the end of the incubation, a Real Time RT-PCR analysis of the expression of different genes was performed. (F) U87MG-CSCs were treated with TNF-α (10 ng/ml)/TGF-β1 (10 ng/ml) in the absence or the presence of CAR (20 µM) in NSC medium for 7 days, then a real Time RT-PCR analysis of the transcription factors (Snail, Slug, Twist and ZEB1) was performed. (G) U87MG cells were treated as indicated, and the levels of miR-200c were quantified at the end of the incubation. The data are presented as the means of three different experiments. The significance of the differences was determined by one-way ANOVA, followed by Bonferroni’s post hoc test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs. the control; ^#P ≤ 0.05, ^##P ≤ 0.01, ^###P ≤ 0.001 vs. TNF-α/TGF-β1 alone; ^§§P ≤ 0.01, ^§§§P ≤ 0.001 vs. CAR alone.