Dual inhibition contributes to down modulation of PI3K/Akt, ERK1/2 and Jak/STAT3 pathways in GBM CSC. (A) Western blots in Case 1 reveal none modulation of Notch1 target Hes1 protein by GSI-X. EGFR inhibition affects the phosphorylation status either of the full-lenght or EGFRvIII. p-Akt1 (S473) and p-ERK1/2 (T202/Y204) disappear with the dual treatment in both of pools, but p-Stat3 (Y705) and PDGFRβ expression was maintained high in p-CSC1 after treatment, and probably contribute to drug resistance. (B) Western blot analysis reveal no modulation of Notch1 target Hes1 protein in both pools of Case 2. Decrease of p-Akt1 protein levels by GSI-X in c-CSC2 but not in p-CSC2. Either p-Erk1/2 or p-Akt1 disappear following dual treatment. Higher levels of p-Stat3 in p-CSC2 compared to c-CSC2 may contribute to drug resistance as observed in p-CSC1. AG1478 alone or combined with GSI-X downmodulates the levels of p-Stat3 and PDGFRβ. (C) Western blot analysis detects significant differences in NICD1, EGFR and PDGFRβ protein expression between core- and p-CSC3. c-CSC3 show no modulation of p-Akt1 and p-Erk1/2 by Notch inhibition, while AG1478 causes p-Erk1/2 decrease but not p-Akt1. Dual treatment triggers a decrease of p-Stat3 with a significant loss of p-Akt1 in c-CSC3. On the contrary, p-CSC3 pool maintains an high expression of p-Akt1, p-Erk1/2, PDGFRβ and p-Stat3 either after AG1478 or dual treatment, which may explain the high cell drug resistance. Arrows denote the doublet of EGFR full-length and EGFRvIII.