Figure 3. Cancer stem cells and differentiation markers ‘expression were evaluated by flow cytometry. (N=3). Chloroquine, Beclin1 and ATG5 protein inhibition significantly increase stem cell markers’ expression in U87-MG but do not significantly modify the expression of differentiation markers except for GFAP (* p<0.05). Cells were cultured for 96 h in normoxic or hypoxic conditions and were treated, or not, with 40 μM chloroquine (CQ) for 24 h, as previously described (32). In basal conditions, the percentage of pLKO cells expressing cancer stem cells markers is close to zero for both Oct4 and CD133. The two stem cells markers increased significantly compared to basal condition after inhibition of autophagy using Chloroquine (+ CQ, 4% ** and $$ p<0.01); in shBeclin1 cells (3% for Oct4 and 10% for CD133 * p<0.05 and $$$ p<0.001 respectively); in shATG5 cells (only for Oct4 *** p<0.001). Concerning differentiated markers no significant results are observed except for GFAP expression that increases after chloroquine treatment.