Figure 4.

PTC-209 inhibits the self-renewal of glioblastoma stem cells (GSCs) in vitro. (A) After treatment of with PTC-209 (1 μM and 10 μM) or DMSO for 4 days, U87MG and T98G cells were respectively incubated with Hoechst 33,342 dye and side population (SP) was determined by FACS. (B) Percentage of SP cells in U87MG and T98G adherent cells in designated groups (mean ± SD; n = 3; **p < 0.01; ***p < 0.001). (C) Following treatment with PTC-209 (1 μM and 10 μM) or DMSO for 4 days, T98G cells were stained with CD133 antibody and subjected to FACS analysis. (D) Percentage of CD133+ cells in T98G adherent cells (mean ± SD; n = 3; ***p < 0.001). (E-F) Extreme limiting dilution analysis was used to measure self-renewal capability of GICs derived from parental adhesive cells under indicated condition. The frequency of sphere-initiating cells and the log fraction nonresponding calculated by webtool at http://bioinf.wehi.edu.au/software/elda/with 95% confidence interval were shown in figure E and F, respectively. The data value with zero negative response at dose 100 is represented by a down-pointing triangle in figure F. (G) Microscopic observation of T98G sphere colonies after 2 weeks under treatment with DMSO, 1 μM PTC-209, 10 μM PTC-209 or 200 μM TMZ. Images are represented for 60 independent wells. Scale bar 100 μm.