Figure 5: U87 xenograft mice treated with II-B08 demonstrate reduced tumour burden.

(a) U87 astrocytes were serum starved and pretreated with (+) or without (−) increasing concentrations of II-B08 followed by treatment with (+) or without (−) EGF lysed and a pull down (PD) assay was conducted using Raf:RBD beads. (b) U87 astrocytes transfected with the indicated plasmids were treated with or without (dimethylsulphoxide (DMSO)) increasing concentration of II-B08 for 18 h, lysed and immunoblotted with the indicated antibodies. Equal number of U87 astrocytes transfected with indicated plasmids were plated in 96-well plates in sextuplicate, treated with or without (DMSO) increasing concentration of II-B08 for 18 h, and alamar blue (c) or 5-bromodeoxyuridine (BrdU; d) assay was then performed. Data represent mean±s.e.m. of three independent experiments performed in sextuplicates. *P<0.05 Student's t-test compared with DMSO control. (e) Equal number of U87 or U87 astrocytes stably expressing EGFRviii deletion mutant (U87-viii) were plated in 96-well plates in sextuplicate, treated with or without (DMSO) increasing concentration of II-B08 for 18 h, alamar blue assay was performed. Data represent mean±s.e.m. of three independent experiments performed in sextuplicates. *P<0.05 Student's t-test compared with DMSO control. (f) Equal number of U87 or U87-viii astrocytes were suspended in agar matrix and treated with or without increasing concentrations of II-B08. Following 10 days incubation, the anchorage-independent growth assay was performed. Data represent mean±s.e.m. of three independent experiments performed in sextuplicates. *P<0.05 Student's t-test compared with DMSO control. Representative images are shown on top panel captured using a Nikon Coolpix 995 digital camera mounted on a VWR Vista Vision inverted microscope. Scale bar, 200 μm. (g) T2 weighted anatomy MRI imaging demonstrated reduced tumour appearance after 7 days of therapy, which when quantified demonstrates a very significant decrease in the tumour volumetrics when compared with placebo (DMSO)-treated control mice following delayed therapy, beginning at 14 days post injection (**P = 0.0053, Student's t-test) and immediate therapy, beginning at the day of injection (**P = 0.0059, Student's t-test). Eight- to ten-week-old mice were used with n = 11 for delayed treatment group, n = 7 for delayed control group, n = 9 for immediate treatment group and n = 7 for immediate control group. Scale bar, 2 mm. (h) Right hemisphere containing the tumour was lysed from mouse treated with or without II-B08 and immunoprecipitated with anti-Ras or control IgG antibody and immunoblotted using the indicated antibodies. (i) Immunohistochemical staining of U87 tumour tissue following immediate and delayed Il-B08 treatments confirms a reduction in SHP2 and Ki67, whereas TdT-mediated dUTP nick end labelling (TUNEL), an apoptotic indicator, remained similar. Images are representative from untreated or immediate or delayed treated mice with II-B08. (j) Quantification of Ki67 as a % of total cells in the field demonstrates a statistically significant reduction following both treatments (***P<0.0001 ANOVA-post-Tukey). (k) Kaplan–Meier survival curve was plotted for control, immediate and delayed Il-B08-treated mice. The immunoblot data are representative of at least three separate experiments. IP, immunoprecipitation; WCE, whole-cell extract.