Research Paper Volume 13, Issue 14 pp 18106—18130

Autophagy inhibition reinforces stemness together with exit from dormancy of polydisperse glioblastoma stem cells

Impact of Beclin1 extinction on glioma cell proliferation and clonogenicity. (A) Cell proliferation in normoxic (N) and hypoxic (H) conditions. Cell proliferation was analyzed using BrdU incorporation (N=3). A slight increase in proliferation rate of U87hBeclin1 cells was observed, as compared with U87pLKO in normoxia; in hypoxic conditions a more significant difference was recorded **pB) Ki67 expression significantly increases in shBeclin1 and shATG5 cells compared to pLKO condition ***pC) Colony forming unit assay with U87shBeclin1. The clonogenic assay was performed as described in material and methods (N=3). There is a significant increase in number (left panel) and size (right panel) of U87shBeclin1 colonies as compared with those obtained with U87pLKO *** pD) Impact of TMZ on cell proliferation. In basal condition, after 144 h of cell culture and 3 h of BrdU incorporation, U87shBeclin1 cells proliferate more than U87pLKO (***pE) Impact of TMZ on apoptosis. Apoptotic cell death was evaluated using the Elisa Cell death kit as described in material and methods. After 144 h of culture in basal condition, U87pLKO appears to be the most sensitive cell line to basal apoptosis compared to U87shBeclin1 (***p

Figure 4. Impact of Beclin1 extinction on glioma cell proliferation and clonogenicity. (A) Cell proliferation in normoxic (N) and hypoxic (H) conditions. Cell proliferation was analyzed using BrdU incorporation (N=3). A slight increase in proliferation rate of U87hBeclin1 cells was observed, as compared with U87pLKO in normoxia; in hypoxic conditions a more significant difference was recorded **p<0.01. (B) Ki67 expression significantly increases in shBeclin1 and shATG5 cells compared to pLKO condition ***p<0,001 (C) Colony forming unit assay with U87shBeclin1. The clonogenic assay was performed as described in material and methods (N=3). There is a significant increase in number (left panel) and size (right panel) of U87shBeclin1 colonies as compared with those obtained with U87pLKO *** p<0.001. (D) Impact of TMZ on cell proliferation. In basal condition, after 144 h of cell culture and 3 h of BrdU incorporation, U87shBeclin1 cells proliferate more than U87pLKO (***p<0.001). TMZ treatment at 1.5 mM induces a very significant decrease in cell proliferation of the two cell lines (*** p<0.001) compared to non-treated cells. There was no difference in proliferative rates between U87pLKO and U87shBeclin1 cells when treated with TMZ (N=3). (E) Impact of TMZ on apoptosis. Apoptotic cell death was evaluated using the Elisa Cell death kit as described in material and methods. After 144 h of culture in basal condition, U87pLKO appears to be the most sensitive cell line to basal apoptosis compared to U87shBeclin1 (***p<0.001). TMZ treatment induces a significant increase in cell apoptosis of U87pLKO cell line (**p<0.01) whereas U87shBeclin1 cells appear to be resistant to TMZ-induced apoptosis (N=3).