Analysis of the differential effect of n-Butanol, U0126, Wortmannin and LY294002 on reporter gene expression and phosphorylation of mTOR in transfected PC3 and 293T cells.

<p>Effect of n-butanol on expression of (<b>A</b>) GFP; (<b>B</b>) β-Gal, and (<b>C</b>) Inhibition of phosphorylation of mTOR by n-butanol; Effect of U0126 (U0), Wortmannin (Wort) and LY294002 (LY) on (<b>D</b>) β-Gal and (<b>E</b>) GFP expression. Proteins were separated by 12–14% SDS-PAGE and detected by immunoblotting using antibodies specific to GFP and β-Gal.</p

Analysis of RNA and protein levels derived from the transfected expression vectors of PDGF-B, GFP and β-Gal in PC3, PC3BM, HeLa, 293T and MA104 cells.

<p>(<b>A</b>) PDGF-B mRNA levels were determined by RNase protection Assay. The 144 nt protected band corresponds to PDGF-B and the 120 nt band represents that of β-Actin mRNA. (<b>B</b>) RT-PCR of β-Gal, GFP and β-Actin mRNA in pcDNA3-β-Gal and pcDNA3-GFP transfected cells. (<b>C</b>) Radioimmunoprecipitation of PDGF-B protein expressed in pCMV-PDGF-B transfected cells using an N-terminal antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014408#pone.0014408-Rao2" target="_blank">[69]</a>. (<b>D</b>) Levels of β-Gal, GFP and β-Tubulin proteins in pcDNA3- β-Gal and -GFP transfected cells. 50 µg of transfected cell lysate was analyzed for GFP and β-Gal levels by SDS-PAGE. (<b>E</b>) β-galactosidase assay using the β-Gal ELISA Kit from Roche Diagnostics. (<b>F</b>) Fluorescence microscopy and bright field (BF) images of 293T, PC3BM, HeLa and PC3 cells transfected with pcDNA-GFP reporter gene construct. (<b>G</b>) Analysis of the fold differences in expression of the reporter mRNA and protein levels between PC3 and HeLa, and PC3 and PC3BM.</p

Western blot analysis of key target proteins of mTOR pathway.

<p>Analysis of total protein and/or phosphorylated forms of (<b>A</b>) mTOR and TSC2, (<b>B</b>) eIF4E, (<b>C</b>) 4EBP1, (<b>D</b>) S6, (<b>E</b>) eEF2 and eEF2K (<b>F</b>) p70S6K, (<b>G</b>) PI3K and PDK1, (<b>H</b>) Akt and (<b>I</b>) ERK1/2, p38 MAPK and PKC. 50 µg of cell lysate was used for analysis of mTOR, TSC2, PI3K, PDK1, Akt, eEF2, 4EBP1 and S6 and 100 µg was used for detection of phosphorylated forms and other proteins.</p

Comparative analysis of the translational regulatory proteins and different signaling pathway proteins in PC3 and 293 T cells.

<p>(<b>A</b>) Analysis of mTOR target proteins 4EBP1, S6, eEF2K and p70S6K. (<b>B</b>) mTOR and TSC2. (<b>C</b>) PI3K/Akt and ERK1/2. (<b>D</b>) PKC. 200 µg of cell lysate was used for PKC analysis and 50 µg was used for analysis of other proteins.</p

DenHunt - A Comprehensive Database of the Intricate Network of Dengue-Human Interactions

<div><p>Dengue virus (DENV) is a human pathogen and its etiology has been widely established. There are many interactions between DENV and human proteins that have been reported in literature. However, no publicly accessible resource for efficiently retrieving the information is yet available. In this study, we mined all publicly available dengue–human interactions that have been reported in the literature into a database called DenHunt. We retrieved 682 direct interactions of human proteins with dengue viral components, 382 indirect interactions and 4120 differentially expressed human genes in dengue infected cell lines and patients. We have illustrated the importance of DenHunt by mapping the dengue–human interactions on to the host interactome and observed that the virus targets multiple host functional complexes of important cellular processes such as metabolism, immune system and signaling pathways suggesting a potential role of these interactions in viral pathogenesis. We also observed that 7 percent of the dengue virus interacting human proteins are also associated with other infectious and non-infectious diseases. Finally, the understanding that comes from such analyses could be used to design better strategies to counteract the diseases caused by dengue virus. The whole dataset has been catalogued in a searchable database, called DenHunt (<a href="http://proline.biochem.iisc.ernet.in/DenHunt/" target="_blank">http://proline.biochem.iisc.ernet.in/DenHunt/</a>).</p></div

Enrichment of KEGG pathways for the dengue virus interacting human proteins.

<p>Pathway enrichment analysis for dengue virus interacting proteins was carried out using WebGestalt. Only pathways that have 3 and more dengue virus interacting proteins and an adj p-value of ≤ 0.01 are selected. The KEGG pathways that were enriched were grouped into broad categories as mentioned in the KEGG pathway database. (a) The broad categories and their total number of dengue virus interacting human proteins are plotted as a pie chart. The color code for each category is given below the pie chart. Pathways that have 20 and more dengue virus interacting proteins belonging to the top 4 broad categories are plotted as bar graph where (b) represents immune system pathways, (c) represents signal transduction pathways, (d) represents transport and catabolism and (e) represents metabolic pathways. In all cases numbers indicates the number of genes identified in each pathway.</p

The number of human proteins involved in direct interactions with N number of dengue viral proteins.

<p>The number of human proteins that can interact with N = 1–7 viral components along with the percentages they constitute are shown in the table.</p

Consolidated list of different types of dengue—human interactions available at the DenHunt database.

<p>The type of interactions, the total number of unique interactions for each type of interaction, the number of references that describe each type of interaction and the total number of human proteins involved in all the interactions are given in the table.</p

Representation of dengue viral interacting proteins in NF-κB and RIG-I-like receptor signaling pathway.

<p>The dengue virus interacting proteins are mapped to the KEGG pathways using the “Search&Colour Pathway” tool from the KEGG database. The proteins that are involved in direct interactions are colored orange, indirect interactions are colored in pink, up-regulated proteins are colored red and down-regulated proteins are colored green. The viral components are depicted as blue hexagons. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004965#pntd.0004965.g003" target="_blank">Fig 3a</a> represents NF-κB and 3b RIG-I-like receptor signaling pathway.</p

List of the DVHFs and the number of FDA drugs, infectious diseases and non-infectious diseases associated with them.

<p>This table contains a list of DVHFs, the number FDA approved drugs against the DVHFs which are used to treat other diseases and the number of infectious and non-infectious diseases associated with the DVHFs.</p