Figure 3

PARP inhibition increases TMZ induced double stranded breaks, autophagy and apoptosis in GSCs. A) DSB induction is significantly enhanced by combination therapy. GSCs, MGMT(+) and MGMT(-) were scored for γH2Ax positive cells, as an indicator for DSB induction,16 hrs post-treatment. GS160 results are all significant as compared to non-treated controls. For the other cultures, significance is indicated as described in the following; * indicates p < 0,05 as compared to non-treated controls. # indicates p < 0.05 as compared to monotherapy. R = combination therapy responder, NR = non-responder. B) Apoptosis induction is significantly enhanced by combination therapy in responder GSCs but not in non-responders. Wells were scored for apoptosis positive cells by live imaging. Read out was performed at 48 hrs post-treatment. Apoptotic cells were significantly (p < 0.05) increased after combination therapy as compared to monontherapy in GSC79. For GS160 cells were not affected by TMZ/ABT-888 combination therapy while the positive control (etoposide 0.1 mM) was successfully inducing apoptosis. C) TMZ and ABT-888 induce autophagy in GS79, which is augmented by combination therapy. Autophagosomes were counted per well and compared to non-treated controls. Readout was performed 16 hrs post-treatment. Compared to controls, all treatment conditions were significantly increased p < 0.05. However, combination therapy vs. monotherapy with ABT-888 or TMZ was found not significantly increased (p = 0.3 and p = 0.4). D) Representative images acquired by the Incucyte FLR of GS79 stained for autophagosomes at indicated treatment conditions. Note the accumulation of speckled dots in treated cells, specifically localized in enlarged cytopathic cells. E) Combination therapy enhances autophagic flux as compared to monotherapy in GS79. Western blot 24 hrs post-treatment indicates the accumulation of LC3-II (lipidated isoform) that is indicative for increased autophagic flux. Actin blots serve as loading controls.