Effect of TMZ on cell survival.

<p>Representative images of (A) U87-MG and U–SC, and (B) L0627 morphology after 48 h treatment with vehicle (0.1% DMSO) or 100 µM TMZ (+ TMZ). Images were viewed on a phase contrast microscope, and digital images were acquired (magnification, 10X; scale bar, 100 µm). (C) U87-MG and U–SC, and (D) L0627 cells were exposed to different concentrations of TMZ or vehicle. Cell viability was assessed after 48 h of treatment by MTT assay. Results are expressed as percentage of cell viability with respect to vehicle-treated cells (100%). Data are the mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01 vs vehicle-treated cells. (E) Cell lysates (60 µg of proteins) were analyzed by immunoblotting with anti-MGMT and anti-β-actin antibodies. 98G human glioblastoma cell lysates were used as control. The immunoblottings are representative of one out of three.</p

Biosynthesis and fates of S1P in U87-MG and glioma stem cells.

<p>U87-MG, U–SC and L0627 cells were pulsed with [³H]-sphingosine (Sph) for the indicated periods of time. At the end, cells and media were processed and analyzed as described in “Materials and methods”. Panel A, total incorporated radioactivity and intracellular Sph. Panel B, radioactivity associated to total Sph phosphorylation, total S1P and S1P degradation (³H ₂O). Panel C, radioactivity incorporated into intracellular and extracellular S1P. Data are the mean ± SD of at least three independent experiments.</p

Role of S1P in GSC survival properties.

<p>U–SC (A) and L0627 cells (B) were exposed to 50-200 µM of TMZ alone (♦) or in combination with 4 µM SKI (●). After 48 hours of treatment, cell viability was assessed by the MTT assay. Results are expressed as percentage of cell viability with respect to vehicle-treated cells (♦) or cells treated with SKI alone (●) (100%). Data are the mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01 vs vehicle-treated cells.</p

Extracellular/intracellular S1P ratio in U87-MG and glioma stem cells.

<p>U87-MG and both GSC types were pulsed with 20 nM [³H]-Sph (0.4 µCi/ml) for 30 or 60 minutes. At the end, S1P was extracted from cells and media and analyzed as described in “Materials and methods”. The extracellular/intracellular ratio of S1P-associated radioactivity is shown, as the mean ± SD of at least three independent experiments.</p

Effect of extracellular S1P on GSC resistance to TMZ.

<p>L0627 cells were exposed to 200 nM S1P, 100 µM TMZ, and 4 µM SKI, separately or in combination, as indicated. When used, after 24 hours 200 nM S1P was added again. After 48 hours of treatment, cell viability was measured by the MTT assay. Results are expressed as percentage of viable cells with respect to vehicle-treated ones (100%). Data are the mean ± SD of at least three independent experiments. * p < 0.05; ** p < 0.01 vs vehicle-treated cells.</p

Expression of sphingosine kinases in U87-MG and glioma stem cells.

<p>Cell lysates (40 µg of proteins) were analyzed by immunoblotting with (A) anti-SK1 and anti-GAPDH antibodies, or (B) anti-SK2 and anti-β-actin antibodies. The immunoblottings are representative of one out of three.</p

Characterization of cell models.

<p>(A) Representative images of U87-MG and U–SC morphology. Images were viewed on a contrast phase microscope and digital images were acquired (magnification, 10X; scale bar, 100 µm). (B) Expression of CD133 and nestin, assessed by Real-Time PCR. Results are expressed as fold-change relative to U87-MG. Values are the mean ± SD of three independent experiments. * p < 0.05; ** p <0.01 vs U87-MG cells.</p