Proposed mechanism of HCV NS3 and adenovirus E1A in the Notch-mediated transcription.

<p>The HCV NS3 protein activates both the SRCAP and p400 functions for the Notch-mediated transcription, whereas adenovirus E1A protein activates only the p400 function. Since E1A disrupts the interaction of p300 and p400, enhancement of SRCAP-mediated transcriptional activation of Notch target genes by NS3 is inhibited by the competition with the E1A function. The NS3 function for p400-mediated transcriptional activation is not competitive with the E1A function. Thus an additional expression of NS3 enhances the E1A function for increments in the p400-mediated transcriptional activation.</p

Identification of SRCAP as a host factor which is targeted by HCV NS3.

<p>(A, B) HEK293 cells were transfected with the expression vectors for HA-tagged NS3 and FLAG-tagged SRCAP (A), or the expression vectors for HA-tagged SRCAP and FLAG-tagged NS3 (B). After 48 hrs, the cells were harvested and the whole cell lysates were subjected to the immunoprecipitation assay using anti-FLAG M2 agarose (Sigma) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020718#s4" target="_blank">Materials and Methods</a>. The asterisk (*) indicates the bands which are considered to be degradation products of exogenously expressed SRCAP. (C) HEK293 cells were transfected with expression vectors for FLAG-tagged NS3 and SRCAP proteins. After 24 hrs, the cells were harvested, and were fractionated into nuclear and post-nuclear fractions. Anti-histon H1 and anti-actin antibodies were used as a nuclear control and a loading control, respectively.</p

NS3 binds to the amino acid region 1449–1808 of the p400 protein.

<p>(A) Schematic illustration of domain structure of human SRCAP and p400. The domain structures predicted by using conserved domain database (CDD) are indicated in the figure. HAS, helicase-SANT-associated (HSA) domain; SNF2N, SNF2 family N-terminal domain; DEXDc, DEAD-like helicases superfamily domain; CBPid, CBP interaction domain; NS3 bd, HCV NS3 protein binding domain; HELICc, Helicase superfamily c-terminal domain. (B) Amino acid sequence alignment of the NS3-binding region of SRCAP and its equivalent region in p400. The NS3 binding region of SRCAP (amino acid position: 1639–1990) was aligned with the p400 sequence using CLUSTAL W <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020718#pone.0020718-Thompson1" target="_blank">[62]</a>. Identical amino acids are shown in gray. (C, D) The expression vector for HA-tagged SRCAP (1639–1990) (C) or p400 (1449–1808) (D) was transfected into HEK293 cells together with the expression plasmid for FLAG-tagged NS3. After 24 hrs, the cells were harvested, lysed, and then subjected to the co-immunoprecipitation analysis using anti-FLAG M2 agarose (Sigma).</p

NS3 and SRCAP cooperatively activate Notch-mediated transcription of <i>Hes-1</i> promoter.

<p>(A) Schematic illustration of domain structure of human Notch1 and Notch3. The amino acid positions of the intracellular domains of Nocth1 and Notch3 are indicated in the figure. EGF-like, EGF-like domain; LNR, LIN12/Notch repeats; TM, transmembrane region; RAM, RAM (RBP-jk-associated molecule) domain; NLS, nuclear localization signal; ANK, ankyrin repeat; TAD, transactivation domain; PEST, PEST (proline (P)-, glutamic acid (E)-, serine (S)- and threonine (T)-rich) sequence. (B) Protein expression analysis of FLAG-tagged intracellular (IC) domain of Notch1 and Notch3. The expression vector for FLAG-tagged Notch1 IC or Notch3 IC was transfected into HEK293 cells. After 24 hrs, the whole cell extracts were subjected to the immuno-blotting analysis using anti-FLAG antibody. (C, D) The expression vectors for FLAG-tagged Notch1 IC (C), or Notch3 IC (D) and the expression plasmids for FLAG-tagged NS3 and SRCAP were transfected into Hep3B cells together with reporter constructs, pGL4.20 <i>Hes-1</i> pro and pcDNA3.1 (+) <i>Rluc</i>. After 48 hr, the luciferase activities were quantified by luminometer. The relative Hes-1 promoter activities are expressed in relative luminescence units (RLU) normalized by <i>Renilla</i> luciferase activities. Error bars indicating the standard deviations were calculated from at least three independent experiments.</p

Protease activity of NS3 is not required for activation of Notch-signaling pathway.

<p>(A) Schematic diagram of the NS3 deletion mutant and its protease deficient mutant used in this study. The amino acid residues of the serine protease catalytic triad and the conserved motifs of helicases are indicated in the figure. (B) The expression vector for the HA-tagged N-terminal protease region of NS3 and FLAG-tagged SRCAP were transfected into HEK293 cells. After 48 hrs, the whole extracts from the cells were immunoprecipitated with anti-FLAG agarose (Sigma), and the immunoprecipitated fractions were analyzed by immunoblotting. (C) The expression vector for FLAG-tagged NS3 or the N-terminal portion of NS3 and the expression plasmid for Notch1 IC were transfected into Hep3B cells together with pGL4.20 Hes-1 pro and pcDNA3.1 (+) Rluc. After 48 hrs, the luciferase activities were quantified by luminometer. (D) The EGFP-fused N-terminal protease region of NS3 or its protease deficient mutant was transfected into Hep3B cells together with the Notch1 IC expression vector, pGL4.20 Hes-1 pro and pcDNA3.1 (+) Rluc. After 48 hrs, luciferase activities were measured by luminometer. (E) The expression vectors for full-length HCV polyprotein and for Notch1 IC were transfected into Hep3B cells together with the reporter gene constructs. After 48 hrs, luciferase activities were quantified. The relative <i>Hes-1</i> promoter activities are expressed in RLU normalized by <i>Renilla</i> luciferase activities. Error bars indicating the standard deviations were calculated from at least three independent experiments.</p

High resolution images of pictures shown in Fig 2A and 2B.

High resolution images of pictures shown in Fig 2A and 2B.</p

Oligo-CS effectively inhibits differentiation of RAW264 cells into osteoclasts.

(A) RAW264 cells were grown in differentiation medium for 5 days to differentiate into osteoclasts. The control cells were grown in differentiation medium without sRANKL. Then the cells were subjected to TRAP staining. (B) RAW264 cells were grown in differentiation medium with the indicated concentrations of CS or Oligo-CS for 5 days, and the cells were subjected to TRAP staining. (C) The TRAP positive mature multinuclear cells were counted under a microscope. Error bars indicate standard deviations calculated from at least 19 frames of each group. Asterisks (*) shown in the graph indicate that the difference is statistically significant (p < 0.05) compared to the control.</p

Marker gene mRNA expressions in C2C12 cells grown in adipocyte differentiation medium.

(A) C2C12 cells were grown in adipocyte differentiation medium for a total of 5 days as described in the Materials and Methods. The concentrations of accumulated lipids were measured by the Oil Red O staining method. (B-G) Marker gene mRNA expressions in the C2C12 cells grown in adipocyte differentiation medium were monitored by real-time RT-PCR analysis using the specific primer set for each of the genes. The data indicate relative expressions compared to cells grown in growth medium after normalization with GAPDH mRNA expression. Error bars indicate standard deviations (n = 3). Asterisks (*: p p < 0.001) indicate that the difference is statistically significant between two groups.</p

Specific primer sets used in this study.

Chondroitin sulfate (CS) is a glycosaminoglycan, and CS derived from various animal species is used in drugs and food supplements to alleviate arthralgia. The CS is a high molecular weight compound, and hydrolysis of CS by intestinal microbiota is thought to be required for absorption in mammalians. Chondroitin sulfate oligosaccharides (Oligo-CS) are produced by hydrolysis with subcritical water from CS isolated from a species of skate, Raja pulchra for the improvement of bioavailability. The present study conducted in vitro experiments using murine cell lines, to compare the biological activities of Oligo-CS and high molecular weight CS composed with the similar disaccharide isomer units of D-glucuronic acid and N-acetyl-D-glucosamine (CS-C). The results show that Oligo-CS inhibits osteoclast differentiation of RAW264 cells significantly at lower concentrations than in CS. The cell viability of a myoblast cell line, C2C12 cells, was increased when the cells were grown in a differentiated medium for myotubes with Oligo-CS, where there were no effects on the cell viability in CS. These results suggest that in vitro Oligo-CS exhibits stronger bioactivity than high-molecular weight CS.</div

Raw and analyzed data for the graphs shown in Figs 2C, 3, 4C, 4D, 5, and 6.

Raw and analyzed data for the graphs shown in Figs 2C, 3, 4C, 4D, 5, and 6.</p