Simultaneous targeting of Notch1 and EGFR signaling induces apoptosis in GBM c-CSC2 and c-CSC3 but not in CSC1. (A) No induction of Caspase-3 and PARP-1 fragments cleavage following drug exposure was monitored. AG1478 induces an high expression of cell cycle regulatory protein p27kip in c-CSC1 and p-CSC1. This finding correlates to a strong shifting from S to G0/G1 phase of cell cycle in both pools as observed by Flow cytometry. CycD1 was not detected by immunoblotting. (B) Appearance of Caspase-3 (17-19 KDa fragments) and PARP-1 (116-89 KDa) fragments are monitored in c-CSC2 either in presence of AG1478 or in combination with GSI-X, while in the p-CSC2 this phenomenon is present but at lesser extent. GSI-X treatment induced a modest apoptosis/necrosis effect preferentially in c-CSC2 and did not affect cell cycle distribution. EGFR signaling inhibition induces apoptosis/necrosis only in c-CSC2 pool, while AG1478 induces a consistent shifting to G1/G0 in p-CSC2 respect to c-CSC2, which correlated with higher levels of p27kip. Cyclin D1 protein is down modulated in both cell populations of case 2 by EGFR inhibition, instead the antiapoptotic protein Survivin declines preferentially in c-CSC2 by the dual blockade, suggesting that the combination therapy is more effective in the c-CSC2 than in p-CSC2. (C) GSI-X and AG1478 concurrently added in c-CSC3 resulted in Caspase-3 and PARP-1 fragments cleavage, decrease of both Cyclin D1 and Survivin expression. AG1478 induces a shift to G0/G1 phase in c-CSC3 and the drugs combination potentiates the apoptotic effect in c-CSC3. p-CSC3 are refractory to any drug blockade as reported by flow cytometry and Western blots analysis. Lower arrows indicate the fragments cleavage of Caspase-3 and PARP-1.