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Results: 4

1.
Fig. 1.

Fig. 1. From: Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma.

Cell lines and experimental strategy. (A) EGFRvIII expression levels in retrovirally transfected U87MG cell lines. (B) Western blot of U87MG cell lines expressing titrated levels of EGFRvIII. Cells were serum-starved for 36 h, lysed, and probed for EGFRvIII or phosphotyrosine levels. (C) Outline of MS-based experimental strategy.

Paul H. Huang, et al. Proc Natl Acad Sci U S A. 2007 July 31;104(31):12867-12872.
2.
Fig. 4.

Fig. 4. From: Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma.

Dose–response of U87-H cell line upon treatment with kinase inhibitors or cisplatin. (A) Dose–response of U87-H cells to AG1478, SU1127, or a combination of SU11274 and 5 μM AG1478 over 72 h after 24-h serum starvation. Viability was measured by using the metabolic dye WST-1. Combination treatment significantly enhanced cytotoxicity at 10 μM SU11274 (P < 0.001). (B) Apoptosis measured by caspase 3/7 cleavage upon drug treatment over 24 h after 24-h serum starvation. Concentration of drugs used was 10 μM SU11274, 10 μM AG1478, or a combination of 10 μM SU11274 and 5 μM AG1478. Combination treatment significantly enhanced apoptosis (P < 0.01). (C) Dose–response of U87-H to AG1478, PHA665752, or a combination of PHA665752 and 5 μM AG1478 over 72 h after 24-h serum starvation. Combination treatment significantly enhanced cytotoxicity at 10 μM PHA665752 (P < 0.0001). (D) Viability of U87-H cells in response to a combination treatment of 10 μg/ml cisplatin with either AG1478 or SU11274.

Paul H. Huang, et al. Proc Natl Acad Sci U S A. 2007 July 31;104(31):12867-12872.
3.
Fig. 3.

Fig. 3. From: Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma.

c-Met receptor activation and kinase inhibition. (A) Western blot of specific phosphorylation sites on the c-Met receptor (Y1230/Y1234/Y1235) across the four different cell lines in vitro after 24-h serum starvation. (B) Western blot of c-Met receptor phosphorylation levels of in vivo parental (P), DK, or EGFRvIII high-expressing U87MG-derived xenografts. (C) Western blot of U87-H cell line subjected to 1 h AG1478 dose escalation after 24-h serum starvation. (D) Comparison of the quantification of the phosphorylation levels for c-Met Y1234 upon treatment with either DMSO (control) or 10 μM c-Met kinase inhibitor SU11274 for 1 h after 24-h serum starvation. Two biological replicates were performed and peak areas for iTRAQ marker ions enable quantification of phosphorylation for each condition.

Paul H. Huang, et al. Proc Natl Acad Sci U S A. 2007 July 31;104(31):12867-12872.
4.
Fig. 2.

Fig. 2. From: Quantitative analysis of EGFRvIII cellular signaling networks reveals a combinatorial therapeutic strategy for glioblastoma.

Effect of EGFRvIII receptor levels on downstream signaling networks. (A) Relative quantification of EGFRvIII phosphorylation sites across the four cell lines. Phosphorylation levels are normalized relative to that of the DK cell line. (B) Visualization of the fold change in phosphorylation levels in the canonical EGFR signaling cascades as a function of titrated EGFRvIII levels. (C) Clustering analysis of phosphotyrosine protein networks using self-organizing maps (SOMs). Each column within the matrix components represent the relative phosphorylation level in the -DK, -M, -H, and -SH U87MG cell lines normalized against the U87H cell line. Optimal SOM architecture was a 3 × 3 matrix, because smaller matrices tended to cluster dissimilar phosphorylation profiles. (D) Protein phosphorylation sites found within the highly responsive cluster.

Paul H. Huang, et al. Proc Natl Acad Sci U S A. 2007 July 31;104(31):12867-12872.

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