Enhanced efficacy using rapamycin and Nimotuzumab was observed in EGFR-null cells. IC50 of (A) TMZ, (B) Nimotuzumab, and (C) rapamycin in iNHA cells was determined by subjecting the cells to a range of drug concentrations. Viability of cells was determined by CCK-8 assay. Data are presented as mean ± SEM. (D) Western blot analysis was carried in EGFR-null Gli36 cells to verify the absence of EGFR expression. A431 cells induced with EGF served as positive (+) control for wtEGFR expression. (E) IC50 of TMZ was determined in Gli36 cells (from 0 to 1000 μM) as described previously. (F) Percentage of cell viabilities of Gli36 cells upon single treatment with either rapamycin (0.1 mM) or Nimotuzumab (0.013 mM) and combination treatment of Nimotuzumab and rapamycin. Data are presented as mean ± SEM. Combination groups were compared to individual single drug treatments ***p < 0.001. (G) Western blot analysis of phospho-ERK1/2, total ERK1/2, phospho-AKT and total AKT in Gli36 cells treated with DMSO, TMZ, rapamycin and Nimotuzumab for 24 h. Pan actin served as loading control. The protein expression is normalized to their respective controls in the blots. The numbers below the blot are relative to the respective controls from arbitrary values generated from the MetaVue software, as described in methods.