Fig. 3
Afatinib and TMZ combination decreases migration and invasion of U87MG and U87 EGFRvIII cells. a, b U87MG and U87EGFRvIII cells (2.5 × 104) were incubated with TMZ (25 μM), afatinib (1 μM) or combination of drugs for 48 h. Non-migrated cells in the upper chamber were removed with a cotton swab, and the migrated cells were stained and counted. Representative images are shown (10X magnification). The bar graph shows the mean (± SD) percentage of migrated cells. Experiments were repeated three times and 5 random fields were chosen for quantification (*$ P ≤ 0.05); * significant compared to control; $ significant compared to TMZ. c, d Afatinib alone or in combination with TMZ decreases the invasion of U87EGFRvIII cells. U87MG and U87 EGFRvIII cells (5.0 × 104) were incubated with TMZ (25 μM), afatinib (1 μM) or combination of drugs for 48 h. Non-invaded cells in the upper chamber was removed with a cotton swab, and the invaded cells were stained and counted. The bar graph shows the mean (± SD) percentage of invaded U87MG and U87EGFRvIII cells. Experiments were repeated three times (*$ P ≤ 0.05); *significant compared to control; $ significant compared to TMZ. e, f Afatinib inhibits EGFRvIII-mediated JAK2/STAT3 and FAK signaling. U87EGFRvIII cells were treated with TMZ (25 μM), afatinib (1 μM) or combination for 48 h, and lysates were analyzed for pEGFR (Tyr-1068), pJAK2 (Tyr-1007/1008), pSTAT3 (Tyr-705), pFAK (Tyr-925) and pAKT (Ser-473) by western blot analysis. g EGFRvIII kinase domain mediates FAK (Tyr-925) activation. U87, U87 EGFR WT, U87EGFRvIII and U87EGFRvIII DK cell lysates were analyzed for pFAK (Tyr-925), pFAK (Tyr-576/577) and pFAK (Tyr-397)