Fig. 2
Afatinib inhibits EGFR and EGFRvIII activation and augments G2/M arrest with TMZ. a U87MG, U87EGFR WT and U87EGFRvIII DK cells were incubated with TMZ (25 μM), afatinib (1 μM) or combination of drugs for 48 h, and cell lysates were analyzed for pEGFR (Tyr-1068) by western blot analysis. β-actin serves as a loading control. b U87EGFRvIII cells were treated with TMZ, afatinib, erlotinib or combination of TMZ and afatinib and analyzed for pEGFR (Tyr-1068). c U251 and U251EGFRvIII cells were treated with afatinib, TMZ or combination for 48 h. Cell lysates were analyzed for pEGFR (Tyr-1068) and pAKT (Ser-473) by western blot analysis. d U87EGFRvIII cells were synchronized with double thymidine block and treated with TMZ (25 μM), afatinib (1 μM) or combination of drugs for 48 h. Cells were trypsinized, fixed with 70% ethanol, stained with Telford reagent and analyzed by flow cytometry. e The bar diagram shows the mean (± SD) percentage of distribution of the cells (*$# P ≤ 0.05); * significant compared to control; $ significant compared to TMZ; # significant compared to afatinib (n = 3)