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Selenomethionine Prevents Degeneration Induced by Overexpression of Wild-Type Human -Synuclein during Differentiation of Neuroblastoma Cells

Center for Vitamins and Cancer Research, Department of Radiology, School of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262

View larger version (10K): [in a new window]   Fig. 1. Schematic representation of retroviral vectors used to establish an over-expressed -synuclein (PN-54) NB cell line. PN-1 was used to generate a vector control cell line. For details see Materials and Methods. Abbreviations: LTR-long terminal repeat of murine leukemia virus; CMV-cytomegalovirus immediate early promoter; MCS-multiple cloning sites; Neo®–neomycin resistance gene; -synuclein-human -synuclein.

View larger version (32K): [in a new window]   Fig. 2. A. Western blot analysis of wild-type human -synuclein holoprotein in undifferentiated (U) and differentiated (D) NBP2, NBP2-PN1 and NBP2-PN54 cells. 20 µg total cellular protein, anti--synuclein antibody, and goat anti-rabbit secondary antibody were used for the analysis. Human -synuclein was detected as a 19 kDa protein. M-marker. B. Levels of cyclophilin A (Cyp-A) were determined and used as a loading control. Each experiment was repeated twice and similar results were obtained.

View larger version (16K): [in a new window]   Fig. 3. Growth rates of undifferentiated NB cell lines. For each cell line, 10,000 cells were plated in each well of a 24 well tissue culture plate. Viability of cells was determined at the indicated time points by MTT assay. Each experiment was repeated three times involving 4 samples per group, and error bars indicate SEM.

View larger version (17K): [in a new window]   Fig. 4. Viability of NB cells differentiated with RO20-1724 and PGA1. Cells (20,000 cells/well) were plated in a 24 well tissue culture plate, and 200 µg/ml RO20-1724 and 2 µg/ml PGA1 were added 24 hours later. Viability of NB cells was determined at the indicated times by MTT assay. Each experiment was repeated three times involving 4 samples per group, and error bars indicate SEM.

View larger version (77K): [in a new window]   Fig. 5. Photomicrographs of differentiated NB cells. Cells (20,000 cells/well) were plated in a 24 well tissue culture plate and treated 24 hours later as described below. NBP2 (A), NBP2-PN1 (B), and NBP2-PN54-C20 (C) cells differentiated for 3 days with 200 µg/ml RO20-1724 and 2 µg/ml PGA1. D. NBP2-PN54-C20 cells differentiated for 3 days with 200 µg/ml RO20-1724 and 2 mM dibutyryl cAMP. E. NBP2-PN54-C20 cells differentiated for 3 days with 200 µg/ml RO20-1724 and 2 µg/ml PGA1 in the presence of 5 µg/ml selenomethionine.

View larger version (14K): [in a new window]   Fig. 6. Effects of selenomethionine on the viability of NB cells differentiated with RO20-1724 and PGA1. Cells (20,000/well) were plated in a 24 well tissue culture plate and were treated with differentiating agents 24 hours after plating. Various concentrations of selenomethionine were added concomitantly with the differentiating agents. Cell viability was measured 3 days after treatment by MTT assay. Each experiment was repeated three times involving 4 samples per group, and error bars indicate SEM.