Journal: Molecular Systems Biology

Article Title: Network modeling of the transcriptional effects of copy number aberrations in glioblastoma

doi: 10.1038/msb.2011.17

Figure Lengend Snippet: Hubs in the CNA-driven glioblastoma network model–based on 10 672 genes and 186 patients

Article Snippet: The duration of PDGF-AA and STI-571 treatment was 14 h. To perturb the EGFR node in U-87MG, U-343MG, U-373MG, T98G and A172, EGFR inhibitor Gefitinib (Selleck Chemicals) and EGFR ligand EGF (supplied by Peprotech) was added at 5 μM and 50 ng/ml, respectively.

Techniques: Binding Assay

Journal: Molecular Systems Biology

Article Title: Network modeling of the transcriptional effects of copy number aberrations in glioblastoma

doi: 10.1038/msb.2011.17

Figure Lengend Snippet: Experimental perturbations of a network region controlled by NDN and PDGFRA . ( A – D ) NDN overexpression slows the growth of glioblastoma cell lines. ( A ) Interactions in the network around EGFR , NDN and PDGFRA . ( B ) Perturbation of NDN by stable overexpression in two separate U343-derived cell lines, denoted as NDN + (moderate overexpression) and NDN ++ (high overexpression). ( C ) Growth curves collected during 6 days showed that NDN overexpression inhibits growth of U343 cells. Error bars indicate 95% confidence intervals. ( D ) Single-time point (7 days) measurement of cell number in NDN -overexpressing cells. Error bars indicate s.e.m. ( E ) Perturbation of PDGFRA by PDGF-AA protein (ligand) and imatinib (STI-571; Gleevec™; inhibits PDGFRA and certain other tyrosine kinases), respectively, produces opposite responses in target genes KCNH8 and FGF9 , which were identified as downstream targets of PDGFRA in the model. NDN overexpression induces CPNE8 target genes and modulates FGF9 response to PDGFRA . Error bars indicate 95% confidence intervals of mRNA expression log 2 -relative to untreated controls. ( F ) Perturbation of EGFR by its ligand EGF and gefitinib (ZD-1839 Iressa™; inhibits EGFR ) produces opposite responses in the predicted EGFR target genes SOCS2 and NR2E1 .

Article Snippet: The duration of PDGF-AA and STI-571 treatment was 14 h. To perturb the EGFR node in U-87MG, U-343MG, U-373MG, T98G and A172, EGFR inhibitor Gefitinib (Selleck Chemicals) and EGFR ligand EGF (supplied by Peprotech) was added at 5 μM and 50 ng/ml, respectively.

Techniques: Over Expression, Derivative Assay, Expressing

Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: (A i) SDS-PAGE analysis showing the purity of the Ni-NTA purified Z EGFR 1907’ under pseudo native and denatured conditions. Pseudo native gel bands (boxed) were extracted and analysed by mass spectrometry. Mass spectrometry data confirmed that both bands correspond to one species, (A ii) DNA sequencing of the plasmid revealed the above sequence and this was also confirmed by mass spectrometry analysis (B i) Elution profile of the size exclusion gel filtration chromatography of Z EGFR 1907’ affibody purified via His-Tag Ni-NTA purification. Superdex 75 1660 column equilibrated with 20 mM Hepes pH 7.4, 500 mM NaCl, 10% Glycerol and 2 mM β mercaptoethanol and 1 ml fractions were collected. Insert shows the complete elution profile. (B ii) 20 µl of selected sample fractions from the purification were denatured, run on an SDS-PAGE gel and stained with Coomassie blue. (C) Silver stained SDS-PAGE gel of the concentrated 1:1 triconjugate Z EGFR 1907’ (PPEA) under pseudo native and denatured conditions. Pseudo native and denatured conditions are indicated by -/+ β-mercaptoethanol.

Article Snippet: Many common cancers over-express the EGFR, by attaching polyIC to PEG and the poly-imine there is less non-specific cellular uptake; the addition of the high affinity EGFR ligand (i.e., the affibody) then directs the polyIC to the tumor cells.

Techniques: SDS Page, Purification, Mass Spectrometry, DNA Sequencing, Plasmid Preparation, Sequencing, Filtration, Chromatography, Staining

Journal: PLOS One

Article Title: EGFR-targeted affibody–polyIC polyplex kills EGFR-overexpressing cancer cells without activating the EGFR

doi: 10.1371/journal.pone.0334584

Figure Lengend Snippet: (A) PPEA-polyplexes selectively kill cell lines overexpressing EGFR. Cells were seeded in duplicates into 96-well plates at a density of 5000 cells in 0.1 ml medium per well and grown overnight. Cells were then treated with polyIC at the indicated concentrations using the PPEA complex. PEI-PEG ratio = 1:1; w/w ratio PEI: polyIC = 0.78. U138MG cells do not express EGFR; U87MGwtEGFR cells express 1x10 6 , A431 express 2-3x10 6 and MDA-MB-468 express 2x10 6 EGFRs/cell. (B,C) In vitro anti-tumor activity of PPEA-polyIC-polyplex is polyIC-specific. A431 and U87MGwtEGFR cell lines were treated with same doses of PPEA-polyIC-polyplex (B) or PPEA polyI polyplex (C) , which served as negative control. Viability was measured by the PrestoBlue Cell Viability Reagent (Invitrogen), according to the manufacturer’s instructions, at 72 hrs after treatment. These experiments were repeated three times with a representative experiment shown.

Article Snippet: Many common cancers over-express the EGFR, by attaching polyIC to PEG and the poly-imine there is less non-specific cellular uptake; the addition of the high affinity EGFR ligand (i.e., the affibody) then directs the polyIC to the tumor cells.

Techniques: In Vitro, Activity Assay, Negative Control