Figure 1. SLM exerts a potent anti-glioma effect in vitro and reduces GSC self-renewal capacity.

(A) Cells were seeded at a density of 5·10³ cells per well in 96-well plates. The following day, cells were incubated with either TMZ or SLM at a concentration ranging from 10⁻³ M to 10⁻⁸ M. Seven days after treatment, cell viability was assessed using MTT assays. The results are expressed as mean values ± SD from three independent experiments and are represented as cell viability relative to non-treated cells (whose viability was taken to be 100%). (B) Median-effect doses (IC50s) of SLM in attached cell lines and neurosphere culture. An IC50 is the median-effect dose (the dose causing 50% of cells to be affected, which is equivalent to 50% survival). The results are expressed as mean values from Figure 1A. (C) GSC11 BTSCs were treated with TMZ or SLM at the indicated concentrations. The number of secondary spheres generated was assessed after 10 days and expressed as relative to non-treated cells (= 100%). To confirm that the spheroids were formed by stem cells, we randomly selected at least 15 individual secondary spheres and subjected them to further, long-term (two-months), propagation in each subcloning experiment. (D) Expression of different stem-cell markers by Q-RT-PCR in GSC11 cells treated with TMZ or SLM. RNA was extracted 72 hrs after treatment and Q-PCR analysis was performed. GAPDH was used as an internal control. To determine relative gene expression, we used the comparative threshold cycle method.