Figure 6. Biological and functional characterization of SdFFF-sorted cells. (A) Cancer stem cell markers expressions. CSCs markers’ expressions are compared to those observed in pLKO using RQ (2-∆∆Ct). POU5F1 and LGR5 gene expressions were significantly increased in F3 U87shBeclin1 cells compared with cells from F1 (respectively **p<0.01 and * p<0.05 for POU5F1 and LGR5) (N=4). (B) Colony forming unit assay Left panel: The clonogenic assay was performed similarly to Figure 3B. Cells from the U87shBeclin1 F3 fraction appear significantly more clonogenic than cells from the U87pLKO F3 fraction. Mid panel: cells from the U87pLKO and U87shBeclin1 F3 fractions present a significant increase in colony number compared with colony number obtained from their F1 counterparts. * p<0.05 and ***p<0.001. In addition, colony number from U87shBeclin1 F3 is significantly higher than colony number from U87pLKO F3 ***p<0.001. Right panel: The size of U87shBeclin1 F3 colonies is significantly higher than the size of U87ShBeclin1 F1 colonies ** p<0.01. U87shBeclin1 F3 colony areas are significantly larger than areas of U87pLKO F3 colonies ***p<0.001. (C) TMZ impact on SdFFF-sorted sub-populations (N=3). Left panel: cell proliferation was examined after 48 h of 1.5 mM TMZ treatment using BrdU incorporation. U87pLKO cells from F3 fraction presented a significantly decreased proliferation rate compared with the corresponding F1 cells ***p<0.001. Cell proliferation of F3 U87shBeclin1 F3 cells was significantly increased as compared with corresponding F1 cells **p<0.01. Right panel: cell death was evaluated by Elisa cell death after 48 h of TMZ treatment at 1.5 mM. Apoptosis induced by TMZ in cells from F3 fractions was significantly more important in U87pLKO cells as compared with U87shBeclin1 cells *** p<0.001. Cell apoptosis in cells from F1 fractions was significantly more important in U87pLKO cells as compared with shBeclin1 cells ***p<0.001 (N=3).