Figure 6. Effect of NHI-2 on the sphere-derived cell morphology.

The GSCs were treated with complete NSC medium containing DMSO (control) or the indicated concentrations of NHI-2 for 7 days. The drug-containing media were replaced with fresh drug-free NSC medium at the end of the treatments, and cells were cultured for an additional 7 days. (a) Representative cell micrographs. (b) The area occupied by the spheres and (c) the length of cellular processes were scored after 7 days of treatment and after 7 days of drug wash-out. The counts represent the mean values ± SEM of three independent experiments. The significance of differences was determined by one-way ANOVA and Bonferroni’s post hoc test: **P ≤ 0.01, ***P ≤ 0.001 vs. the control; ###P ≤ 0.001 vs. the GSCs treated for 7 days without wash out. (d) The GSCs were treated with complete NSC medium containing DMSO (control) or the indicated concentrations of NHI-2 for 7 days. At the end of treatments, the relative mRNA levels of CD133, MAP2 and GFAP were quantified by real time RT-PCR. The data were expressed as the fold change vs. the levels of the control, which were set to 1, and are the mean values ± SEM of three different experiments. *P ≤ 0.05, ***P ≤ 0.001 vs. the control. (e,f) The GSCs were treated for 7 days with complete NSC medium containing DMSO (control) or NHI-1 or NHI-2 (10 μM). At the end of treatments, the obtained cell lysates were subjected to Western blotting. (e) Representative Western blots. The full-length blots of the cropped images shown in the main Figures are included in the Supplemental Information. (f) The bar graph shows the results of the quantitative analysis of the Western blots, which was performed using the ImageJ program. The data were expressed as the percent change in the optical density of the immunoreactive band relative to that of the control, which was set to 100%, and are the mean values ± SEM of three different experiments. **P ≤ 0.01, ***P ≤ 0.001 vs. the control.