Figure 4. Rapamycin and Triciribine abolish sphere-forming ability of human neuronal and glial cancer cell lines.

(A) Representative bright-field images of U251 and SH-SY5Y spheres with or without Rapamycin and Triciribine treatment. Images were visualized by Axiovert inverted microscope at 10× magnification and analyzed by Carl Zeiss Zen 2012 image software. (B) Quantification of the average size of U251 and SH-SY5Y spheres with or without treatment conditions. Data represent an average area (μm²) of 20–30 measured spheres. The data are reported as mean ± SEM (p < 0.001; One-way ANOVA; ^***P < 0.001; different treatment concentrations compared to control, Tukey’s multiple comparison test). (C) Increasing the concentration of treatment of both Rapamycin and Triciribine on U251 and SH-SY5Y spheres, cultured in Matrigel™, led to a similar decrease in the number of sphere-forming units (SFU) in both cell lines. The generated spheres are referred to as G1 (Generation 1) spheres. Results are expressed as SFU which is calculated according to the following formula: SFU = (number of spheres counted × number of input cells) × 100. Data represent an average of three independent experiments. The data are reported as mean ± SEM (p < 0.001; One-way ANOVA; ^***P < 0.001; different treatment concentrations compared to control, Tukey’s multiple comparison test).