Journal: PLoS ONE

Article Title: JC Virus Mediates Invasion and Migration in Colorectal Metastasis

doi: 10.1371/journal.pone.0008146

Figure Lengend Snippet: (A) An illustration of the JCV early transcript region that codes for 5 early transforming proteins, T-Ag, t-Ag and the 3 splice variants T' 165 , T' 136 , T' 135 . T-Ag is predominant protein expressed after transfection (marked in red). (B) RT-PCR and Western immunoblotting gel images depicting JCV T-Ag specific mRNA and protein expression in stably transfected cells (indicated as T-Ag), while no T-Ag expression was observed in control cell lines (V, vector transfected). GAPDH (RT-PCR) and β-actin (WB) were used as loading controls. (C) Immunofluorescence staining with JCV T-Ag antibody shows nuclear expression of JCV T-Ag in transfected HCT116 and SW837 cells lines. The images were taken at a final magnification of 630×.

Article Snippet: Two human CRC cell lines, HCT116 (microsatellite instability or MSI) and SW837 (microsatellite stable or MSS) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Stable Transfection, Control, Plasmid Preparation, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: JC Virus Mediates Invasion and Migration in Colorectal Metastasis

doi: 10.1371/journal.pone.0008146

Figure Lengend Snippet: Migration and invasion assays were performed in Boyden chambers without and with Matrigel respectively. Representative images from one of the three independent experiments showing migration (A) and invasion (B) in control and T-Ag transfected HCT116 and SW837 cell lines (200× magnification). The bar graphs in panels B and D are quantitative determinations of data obtained using ImageJ cell counter software from 3 independent experiments. As indicated, T-Ag transfection (red and blue bars) showed significantly increased migration and invasion ( p<0.05 ) in both cell lines.

Article Snippet: Two human CRC cell lines, HCT116 (microsatellite instability or MSI) and SW837 (microsatellite stable or MSS) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Migration, Control, Transfection, Software

Journal: PLoS ONE

Article Title: JC Virus Mediates Invasion and Migration in Colorectal Metastasis

doi: 10.1371/journal.pone.0008146

Figure Lengend Snippet: (A) The flow chart represents the strategy used for gene expression analysis. Using unsupervised analysis we selected 5559 genes. Dendograms in panels B and C illustrate clustering analysis of JCV-T-Ag transfected cells compared to vector controls. Gene trees are represented on the horizontal axis, while condition trees are represented on the vertical axis. The color conventions for all maps are as follows: red indicates over-expressed transcripts, blue are under-expressed transcripts, and yellow indicates transcripts that did not deviate from the controls. Clustering analysis in panel B illustrates Ingenuity pathway analysis which revealed 529 genes that are involved in regulation of cell motility. Of these, a subset of 43 genes that are specifically involved in migration or invasion and were up- or down-regulated in both cell lines are shown in panels C (as dendogram) and D (Venn diagram). Of this group of 43 genes, 20 genes that are directly or indirectly involved in AKT and MAPK pathways were shared between both HCT116 and SW837 cells as shown in panel E (Venn diagram) and panel F (as a schematic interaction of individual genes with each other). Red indicates up-regulated and green indicates down-regulated genes following T-Ag transfection (panel F).

Article Snippet: Two human CRC cell lines, HCT116 (microsatellite instability or MSI) and SW837 (microsatellite stable or MSS) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Gene Expression, Transfection, Plasmid Preparation, Migration

Journal: PLoS ONE

Article Title: JC Virus Mediates Invasion and Migration in Colorectal Metastasis

doi: 10.1371/journal.pone.0008146

Figure Lengend Snippet: (A) Microarray global gene expression data were validated in a randomly selected subset of genes by quantitative RT-PCR. Data are represented as the mean fold change normalized to vector cells. As indicated, there was a tight correlation between microarray and qRT-PCR in each instance. (B) A subset of cancer-metastasis related genes including MMP-9, uPA, CCL5 and NOS3 showed increased expression following T-Ag transfection in HCT116 cells. (C) Western blotting data indicated increased phosphorylation of AKT as well as ERK1/2 in T-Ag transfected cells in comparison to control cell lines.

Article Snippet: Two human CRC cell lines, HCT116 (microsatellite instability or MSI) and SW837 (microsatellite stable or MSS) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Microarray, Gene Expression, Quantitative RT-PCR, Plasmid Preparation, Expressing, Transfection, Western Blot, Phospho-proteomics, Comparison, Control

Journal: PLoS ONE

Article Title: JC Virus Mediates Invasion and Migration in Colorectal Metastasis

doi: 10.1371/journal.pone.0008146

Figure Lengend Snippet: Migration (panels A and B) and invasion (panels C and D) were performed using Boyden chambers without or with Matrigel. LY294002 (25 µM) and U0126 (10 µM) were added to both upper and lower chambers. Cells that invaded through Matrigel, and migrated through the 8 µm pores in the membrane were stained with DAPI. Cell counting was performed with ImageJ software. Data in bar graphs demonstrate fold changes normalized to vector cells (means±SE) obtained from 3 independent experiments. Significant inhibition in both migration and invasion was observed in both HCT116 and SW837 cells with LY294002 and U0126 individually and in combination.

Article Snippet: Two human CRC cell lines, HCT116 (microsatellite instability or MSI) and SW837 (microsatellite stable or MSS) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Migration, Membrane, Staining, Cell Counting, Software, Plasmid Preparation, Inhibition