Figure 7.

Effect of combined treatments with TMZ and A₁AR or A_2BAR agonists on CSC morphology. (A) CSCs were treated for 7 days with complete NSC medium containing DMSO (a), 100 μM TMZ (b) or TMZ in combination with 100 nM CHA (c), or containing TMZ in combination with 50 nM BAY606583 (d). At the end of treatment periods, the drug-containing media were replaced with fresh drug-free NSC medium, and the cells were cultured for another 7 (a', b', c', d') or 14 (a”, b”, c”, d”) days. Representative micrographs taken after 7 days of treatment and after 7 or 14 days of drug wash-out are shown A. The area of the culture plates occupied by the spheres (B) and the number of spheres (C) were determined after 7 days of treatment and after 7 and 14 days of drug wash-out. The counts are the mean values±S.E.M. of three independent experiments. The significance of the differences was determined using a one-way ANOVA with the Bonferroni post-test: **P<0.05, ***P<0.001 versus control; ^#P<0.05, ^##P<0.01, ^###P<0.001 versus TMZ alone. (D) CSCs were treated as in A and their proliferation was evaluated using the MTS assay. The data were expressed as the percentage relative to that of the untreated cells (control), which was set to 100%, and they are the mean values±S.E.M. of three independent experiments, each performed in duplicate. The significance of the differences was determined using a one-way ANOVA with the Bonferroni post-test: ***P<0.001 versus control; ^§§P<0.01 versus cells treated for seven days