Journal: PLoS ONE

Article Title: Bcl-w Enhances Mesenchymal Changes and Invasiveness of Glioblastoma Cells by Inducing Nuclear Accumulation of β-Catenin

doi: 10.1371/journal.pone.0068030

Figure Lengend Snippet: A , premade brain cancer tissue microarrays (AccuMax, A221-iv) from patients were subjected to immunohistochemical staining with anti-Bcl-w antibody (R & D systems, Minneapolis, MN). Non-neoplastic and corresponding glial tumor grade iii/iv tissues from patients (P1-P12). Bar scale, 100 µm. B , U251 cells were transfected with either empty pcDNA vector or that containing Bcl-w cDNA. Bcl-w expression was detected using Western blotting with β-actin as the loading control. Expression levels of mesenchymal proteins were analyzed using Western blot analysis with anti-Twist1, anti-Snail, anti-Slug, anti-vimentin, anti-E-cadherin and anti-Bcl-w in control vector and Bcl-w-overexpressing cells. C , U251 cells were transfected with control or siRNA oligonucleotides targeting Bcl-w (20nM) for 24 hours. Transfected control or si-Bcl-w U251 cells were subjected to Western blot analysis with the indicated antibodies. D , confocal microscopy analysis of vector or Bcl-w-overexpressing cells showing Bcl-w (Red, Alexa 568) and vimentin (Green, Alexa 488) and DAPI (Blue). Scale bar, 50 µm.

Article Snippet: The U251, U373, U87MG (glioma), MDA-MB-231 (breast cancer) and H1299 (lung cancer) were obtained from the Korean Cell Line Bank (KCLB).

Techniques: Immunohistochemical staining, Staining, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Confocal Microscopy

Journal: PLoS ONE

Article Title: Bcl-w Enhances Mesenchymal Changes and Invasiveness of Glioblastoma Cells by Inducing Nuclear Accumulation of β-Catenin

doi: 10.1371/journal.pone.0068030

Figure Lengend Snippet: Down-regulation of Twist1 and Snail leads to inhibition of glioma invasion and vimentin expression. A , the indicated U251 cell transfectants were incubated in serum-free medium in the presence or absence of PI3K inhibitor (LY294002 (LY); 10 µmol/L) and Akt inhibitor (Akt-I; 10 µmol/L) for 1 hour. Expression levels of Twist1, snail and vimentin proteins were compared using Western blotting. B , control or Bcl-w-expressing U251 cells were transfected with 20nM of Twist1, Snail or vimentin siRNA for 24 hours and were subjected to Western blotting with mesenchymal-related proteins or anti-Bcl-w antibodies. C , cells in incubated in a Matrigel-coated transwell for 20 hours. *, p < 0.05, **, p < 0.005, n = 5.

Article Snippet: The U251, U373, U87MG (glioma), MDA-MB-231 (breast cancer) and H1299 (lung cancer) were obtained from the Korean Cell Line Bank (KCLB).

Techniques: Inhibition, Expressing, Incubation, Western Blot, Control, Transfection

Journal: PLoS ONE

Article Title: Bcl-w Enhances Mesenchymal Changes and Invasiveness of Glioblastoma Cells by Inducing Nuclear Accumulation of β-Catenin

doi: 10.1371/journal.pone.0068030

Figure Lengend Snippet: A , either empty pcDNA vector or that containing Bcl-w cDNA introduced into U251 cells. Bcl-w expression was detected using Western blotting. B , Bcl-w promotes migration and invasiveness of U251 glioblastoma cells. Top, the confluent cells in five fields from the scratched area (200 x 500 µm 2 ) were counted under a light microscope. Transfectants were seeded onto Matrigel-coated polycarbonate filters to analyze their invasive potential. Cells were incubated for 20 hours in modified Boyden chambers, and the number of cells invading through filters stained and counted under a light microscope. Bottom, mean of triplicate experiments significantly different from controls. *, p< 0.05. C , two different siRNA sequences targeting Bcl-w (20nM of si-Bcl-w-1 and si-Bcl-w-2) were introduced into U251 cells for 24 hours, and the invasion assay conducted after 24 hours of incubation. Experiments were repeated five times, and the mean values and standard deviations determined. *, p< 0.05; **, p < 0.005.

Article Snippet: The U251, U373, U87MG (glioma), MDA-MB-231 (breast cancer) and H1299 (lung cancer) were obtained from the Korean Cell Line Bank (KCLB).

Techniques: Plasmid Preparation, Expressing, Western Blot, Migration, Light Microscopy, Incubation, Modification, Staining, Invasion Assay

Journal: PLoS ONE

Article Title: Bcl-w Enhances Mesenchymal Changes and Invasiveness of Glioblastoma Cells by Inducing Nuclear Accumulation of β-Catenin

doi: 10.1371/journal.pone.0068030

Figure Lengend Snippet: A , levels of p-Akt, p-GSK3β, GSK3β, p-β-catenin, β-catenin and TCF-4 in cell lysates were compared by Western blotting using β-actin as a loading control. Conditioned media were prepared by incubating the vector and Bcl-w transfectants in serum-free medium for 24 hours. MMP-2 and MMP-9 activities were compared using zymography. Protein loading volumes were verified with Ponceau S staining. B , levels of β-catenin protein that translocated into the nucleus and Bcl-w protein in vector- or Bcl-w-transfected U251 cells were examined using confocal microscopy. Cells were stained with anti-β-catenin (green) or anti-Bcl-w (red) antibody, followed by nuclear staining with DAPI (blue). Scale bar, 50 µm. C , after separation of cells into cytoplasm and nuclear fractions for the indicated transfectants, each fraction was subjected to Western blotting with anti-β-catenin, anti-Lamin A/C (nucleus marker) and anti-β-actin (cytoplasm marker) antibodies.

Article Snippet: The U251, U373, U87MG (glioma), MDA-MB-231 (breast cancer) and H1299 (lung cancer) were obtained from the Korean Cell Line Bank (KCLB).

Techniques: Western Blot, Control, Plasmid Preparation, Zymography, Staining, Transfection, Confocal Microscopy, Marker

Journal: PLoS ONE

Article Title: Bcl-w Enhances Mesenchymal Changes and Invasiveness of Glioblastoma Cells by Inducing Nuclear Accumulation of β-Catenin

doi: 10.1371/journal.pone.0068030

Figure Lengend Snippet: A , the indicated U251 cell transfectants were incubated in serum-free medium in the presence or absence of PI3K inhibitor (LY294002 (LY); 10 µmol/L) or Akt inhibitor (Akt-I; 10 µmol/L) for 1 hour. Expression levels and activities of p-Akt, p-GSK3β, β-catenin, TCF-4 and MMP-2 proteins were compared using Western blotting. B , cells treated with PI3K inhibitor or Akt inhibitor in the lower compartments of the invasion chambers for 24 hours, respectively. Invasive potential of treated cells was compared. *, p< 0.05 versus untreated control, n = 5. C , β-catenin and TCF-4 siRNAs (20nM) were introduced into vector or Bcl-w overexpressing cells, and cellular levels of β-catenin, TCF-4, MMP-2 and p-FAK compared after 24 hours of incubation using Western blotting with β-actin as a loading control. D , invasive potential of the indicated transfectants was compared. *, p< 0.05, n = 5.

Article Snippet: The U251, U373, U87MG (glioma), MDA-MB-231 (breast cancer) and H1299 (lung cancer) were obtained from the Korean Cell Line Bank (KCLB).

Techniques: Incubation, Expressing, Western Blot, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: Bcl-w Enhances Mesenchymal Changes and Invasiveness of Glioblastoma Cells by Inducing Nuclear Accumulation of β-Catenin

doi: 10.1371/journal.pone.0068030

Figure Lengend Snippet: A , right image, MMP-2 siRNA (20nM) was introduced into the indicated U251 transfectants, and after 24 hours of incubation, p-FAK (Y397) and MMP-2 protein levels were compared using Western blotting. Left image, invasion assays were performed using small interfering RNA MMP-2-treated and untreated cells. *, p< 0.01 versus untreated control, n = 5. B , top image, FAK siRNA was introduced into the indicated transfectants, and after 24 hours of incubation, cellular levels of FAK, p-FAK and MMP-2 compared using Western blotting. Bottom plots, invasion assays were conducted using the indicated cells. *, p< 0.05, n = 5. C , top images, vector- and Bcl-w-expressing cells were transiently transfected with expression vectors for HA-tagged dominant-negative FAK mutant (FAKY397F). After 24 hours of incubation, expression of the introduced mutants in cells was verified by Western blotting. Bottom plots, invasive potentials of the indicated cells were compared. *, p < 0.05, n = 5.

Article Snippet: The U251, U373, U87MG (glioma), MDA-MB-231 (breast cancer) and H1299 (lung cancer) were obtained from the Korean Cell Line Bank (KCLB).

Techniques: Incubation, Western Blot, Small Interfering RNA, Control, Plasmid Preparation, Expressing, Transfection, Dominant Negative Mutation, Mutagenesis