Fig. 2. TMZ sensitivity of EGFRvIII−/+ GBM cells and experimental tumors.

DKMGvIII−/+ and BS153vIII−/+ human GBM cell lines were used for further analysis. a EGFRvIII-specific immunofluorescence staining (red). Cell nuclei were counterstained with DAPI. b EGFRvIII expression was detected by flow cytometry using an EGFRvIII-specific antibody (L8A4). A secondary antibody control was used to assess unspecific staining. c Detection of EGFR, EGFRvIII, and MGMT by western blot analysis. MGMT expressing Jurkat cells (JC) served as a positive and β-actin as loading control. d Analysis of MGMT promoter methylation by PCR. M1, and M2 delineate reactions with methylation-specific primers, U1 with primers for unmethylated DNA. e Analysis of proliferation. DKMGvIII−/+ and BS153vIII−/+ cells were treated 24 h after seeding with 20 µM TMZ. Cell number was determined up to 8 days (mean with S.E.M, n = 5). f Cell growth after 7 days of TMZ treatment. DMSO served as control. The mean value of treated cells was normalized to the mean value of untreated cells (mean with S.E.M, n = 5; P-values are obtained by two-tailed Student’s t test, *p < 0.05, **p < 0.01). g Survival of DKMGvIII−/+ and BS153vIII−/+ cells after TMZ treatment assessed by colony forming assay (mean with S.E.M, n = 4; P-values are obtained by two-tailed Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001). h Relative cell survival of BS153vIII+ after siRNA-mediated EGFRvIII knockdown. An siRNA against cyclophilin B served as a control (mean with S.E.M, n = 4; one-tailed Student’s t test was, *p < 0.05, **p < 0.01, ***p < 0.001). i Xenograft tumor response to TMZ. Two weeks after the intracranial injection of 2.5 × 10⁵ BS153vIII−/+ cells the mice were treated with 5 mg/kg/d TMZ or solvent for five days. Graph: Kaplan–Meier estimates of survival.