EGFRvIII conferred enhanced sensitivity to drug treatment in isogenic human glioma cells. (A) Western blot analysis of EGFR expression in human glioma cells U87MG.EGFRvIII and U87MG.wtEGFR that were engineered to express EGFRvIII and wtEGFR proteins, respectively. Cell viability assay was carried out in (B) U87MG.wtEGFR (C) U87MG.EGFRvIII cells using CCK-8 assay after treatment with 500 μM of TMZ, 0.1 mM of rapamycin or 0.013 mM of Nimotuzumab and combination of rapamycin and Nimotuzumab. Percentage cell viabilities were measured at 24 h after respective treatments. Data are presented as mean ± SEM. Combination group was compared to Nimotuzumab single treatment for each cell line *p < 0.05, **p < 0.01. (D) Western blot analysis was performed to detect the expression levels of phospho-AKT (Ser473) and total AKT in U87MG. wtEGFR and U87MG.EGFRvIII cells untreated (UT) or after treatment with Nimotuzumab (N) for 24 h. Pan actin served as the loading control. Densitometry quantification of the AKT activated levels were determined as described before. The numbers below the blot are displayed as ratio of the total AKT proteins after normalizing with pan actin. (E) U87.wtEGFR cells were treated with TMZ (500 μM), rapamycin (0.1 mM) or Nimotuzumab (0.013 mM and 0.0065 mM) as single treatments and combination of rapamycin and Nimotuzumab for 24 h and percentage cell viabilities were determined by CCK-8 assay. Data are presented as mean ± SEM. Nimotuzumab treatment group at half the concentration (0.0065 mM) was compared to the original group (0.013 mM) in single treatments and in combination with rapamycin ***p < 0.001.