1
On problem with IRES vectors ?inability to transcribe the transcript following the IRES sequence if the first MCS is empty. The new pIRES2-acGFP1acGFP1 vector was used as a control for the pIRES2Gal1-acGFP1 construct. Both vectors were sequenced through their multiple cloning sites to ensure no PCRinduced mutations were present.Transfection and stable clone generationParental, control, and galecti
1
Thanized and their brains removed and either frozen using Optimal Cutting Temperature (OCT) compound (Electron Microscopy Sciences, Fort Washington, PA) in cryomolds placed atop dry ice, or fixed in 10 buffered formalin and paraffin embedded for histopathological analysis. The symptom-free survival of nude mice harboring U87MG parental versus transfected xenografts was compared. Kaplan-Meyer surv
1
Galectin-1 transfectants. A population of GFP-sorted cells (the "Gal-1" bars in Figure 4A) was compared to its parental counterpart. The number of metabolically-active cells attached to fibronectin was no different between the two lines at eight hours. Changing the media at four hours reduced the number of cells left for labeling, but the effect was equal in both groups, suggesting a similar rate
1
By the ample amount of normal mouse brain tissue available for dissection. In spite of species differences, cross-hybridization of mouse genetic material to human probes did prove to be a common occurrence. These data made it possible to control, rather stringently, for the potential contamination of tumor edge samples with mouse brain. Of course, there could still be possible contamination ?react
1
Rbance values of the other wells. Average corrected absorbance was compared between transfectant and parental cells, using a t-test.ECM attachment assaysThe U87MG human glioma cell line was kept in tissue culture in DMEM (Cellgro Mediatech, Inc.), with 10 fetal bovine serum, and penicillin/streptomycin. For transfection, 2.5x106 cells were plated overnight on a 100 mm round dish. Cells were trans
1
Erulescens green fluorescent protein; FACS: Flow-assisted cell sorting; SDS: Sodium dodecyl sulfate; MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2 H-tetrazolium; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; CCD: Charge-coupled device. Competing interests None of the listed authors have competing interests related to the publication of this man
1
Nostics, INC, New Jersey) along with a human-specific mouse monoclonal anti-vimentin antibody (Dakocytomation,Toussaint et al. Molecular Cancer 2012, 11:32 http://www.molecular-cancer.com/content/11/1/Page 4 ofTrappes, France). After incubation with the provided goat anti-mouse secondary antibody, staining developed with NovaRed Developing Reagent (Vector Laboratories, Burlingame, CA). Sections we
1
O the culture media markedly and specifically increased cell migration levels in human neoplastic astrocytes, and that these effects were related to striking modifications in the organization of the actin cytoskeleton and an increase in small GTPase RhoA expression [33]. Conversely, knocking down galectin-1 expression in U87MG GBM cells by stable transfection with antisense galectin-1 mRNA, the co