Rat anti-r<i>Cs</i>HK serum affects <i>C</i>. <i>sinensis</i> adult survival in vitro.

<p>(A) The median survival of <i>C</i>. <i>sinensis</i> adults in the blank control group, 1:40 pre-immune serum group, 1:80 pre-immune serum group, 1:160 pre-immune serum group, 1:40 anti-r<i>Cs</i>HK serum group, 1:80 anti-r<i>Cs</i>HK serum group, and 1:160 anti-r<i>Cs</i>HK serum group was 15, 8, 8, 9, 2, 3, and 3 days, respectively. There was no significant difference in survival rates among pre-immune serum groups at any dilution (<i>p</i> > 0.05). Significant differences were observed in the survival rates among the other groups (<i>p</i> < 0.05). (B) The enzymatic activity of <i>Cs</i>HK in homogenate of parasites collected from each group at 1, 3, 5, and 6 days of incubation. The enzymatic activity of <i>Cs</i>HK in adult worms incubated in medium with different dilutions of anti-r<i>Cs</i>HK serum declined significantly in a dose- and time-dependent manner. (C) As a control, there was no obvious change of the enzymatic activity of <i>Cs</i>PLA₂ in the worms.</p

ELISA of antibody titers and isotype of IgG induced by r<i>Cs</i>HK.

<p>(A) Antibody titers of IgG induced by r<i>Cs</i>HK. (B) IgG isotype induced by r<i>Cs</i>HK. * <i>p</i> < 0.01.</p

Substrate specificity of r<i>Cs</i>HK.

<p>^a from reference [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003641#pntd.0003641.ref017" target="_blank">17</a>].</p><p>Substrate specificity of r<i>Cs</i>HK.</p

Worm burden and EPG of rats in different groups.

<p>Results of analysis represent the mean ± SD, and the recovered worm numbers and EPG in groups were compared by Student’s <i>t</i>-test.</p><p>^a<i>p</i> > 0.05 and</p><p>^b<i>p</i> < 0.01 (compared with PBS group).</p><p>Worm burden and EPG of rats in different groups.</p

Effects of phosphate donors, effectors and EbSe on the enzyme kinetics of r<i>Cs</i>HK.

<p>The effect of 0~3 mM phosphate donors (ATP, CTP, GTP, ITP, TTP, and UTP) and fixed 3 mM glucose (A). The effect of 0~5 mM AMP (B), 0~10 mM PEP (C), 0~10 mM citrate (D), or 0~100 μM EbSe (E) and fixed 3 mM glucose with respect to ATP. The effect of 0~100 μM EbSe and fixed 3 mM ATP with respect to glucose (F).</p

Immunolocalization of <i>Cs</i>HK in <i>C</i>. <i>sinensis</i> and in liver from infected rats.

<p>Mouse anti-r<i>Cs</i>HK serum and anti-mouse IgG were applied as primary antibody and secondary antibody, respectively. Serum from pre-immune mice was employed as primary antibody for a negative control. Panels H, L, P, R, U, V, W, and X are negative controls. Panels B, D, F, H, J, L, N, P, and R are under fluorescence microscope and the same parts (panels A, C, E, G, I, K, M, O, and Q) are under white light. Panels B, D, and F, localization of <i>Cs</i>HK in adult worms; panel J, localization of <i>Cs</i>HK in metacercariae. Panels S and T, localization of <i>Cs</i>HK in intrahepatic bile ducts of a <i>C</i>. <i>sinensis</i> infected rat. In panels S, T, U, V, W, and X, peroxidase staining shows as a yellow/rust colored deposit and Mayer’s hematoxylin counterstains the nuclei in light purple. White arrows highlight the regions of intrahepatic bile duct tissue and the tissue that stained positive for <i>Cs</i>HK. Original magnification: × 50 for panels M, N, O, P, Q and R; × 100 for panels A, B, C, D, E, F, G, H, S, U, and W; × 400 for panels I, J, K, L, T, V, and X. Bar = 800 μm. v, vitellarium; e, egg; vs, ventral sucker; tg, tegument; i, intestine; u, uterus; ts, testicle; o, ovary; p, pharynx; s, spermatheca; l, lumen; w, within the cells; <i>Cs</i>, <i>C</i>. <i>sinensis</i>; BE, biliary epithelium.</p

Additional file 2: Figure S2. of Clonorchis sinensis granulin: identification, immunolocalization, and function in promoting the metastasis of cholangiocarcinoma and hepatocellular carcinoma

Densitometric analysis of genes from Fig. 8a, b, d . Densitometric results were analysed with Image J software. Statistical comparisons between more than two experimental groups were made with one-way ANOVA tests followed by Tukey’s multiple comparisons test. Results are reported as the mean ± standard error of the mean (SEM), and P was set to 0.05. For all analyses, Prism 5.0 software (Graph Pad Software, San Diego, USA) was used. a *P < 0.05, **P < 0.01, compared with the control group. b *P < 0.05, **P < 0.01 and ***P < 0.001, indicate difference from experimental treatment. ## P < 0.01 and ### P < 0.001, compared with the matched pair. (TIF 910 kb

Gene/protein expression analysis of <i>Cs</i>severin at different life cycle stages of <i>C. sinensis</i>.

<p>(A) Quantitative real-time PCR analysis. The transcription levels of <i>Cs</i>severin at life stages of adult worm, metacercaria and egg were analyzed by means of the 2^−ΔΔCt ratio, with <i>Cs</i> β-actin serving as the internal standard. *: <i>p</i><0.05, the transcription level of <i>Cs</i>severin in egg was statistically higher than that in adult worm and metacercaria. (B) Western blotting analysis. Thirty microgram of total proteins from each life cycle stage were subjected to SDS-PAGE and analyzed. Rat anti-r<i>Cs</i>severin serum was used as primary antibody at a dilution of 1∶100. The same dilution of pre-immune rat serum was used as a negative control, and no corresponding band was observed (not shown).</p

Immunolocalization of <i>Cs</i>severin in adult worm and metacercaria of <i>C. sinensis</i>.

<p>Rat anti-r<i>Cs</i>severin serum was used as primary antibody and red fluorescent Cy3-labeled goat anti-rat IgG as secondary antibody. Slides were observed under white light (pane A, C, E, G, I) or fluorescence microscope (pane B, D, F, H, J). No specific fluorescence was observed in pane B or H which was probed with serum from rat immunized with PBS as negative control. Intensive reddish-orange fluorescences were observed in vitellarium and intrauterine eggs of adult worm (pane D, F, ×50) and oral suck of metacercaria (pane J, ×200). Scattered fluorescences were detected in tegument of adult worm and metacercaria. V, vitellarium. O, oral sucker. T, tegument. E, eggs. W, cyst wall. P, pharynx.</p

Additional file 1: Figure S1. of Clonorchis sinensis granulin: identification, immunolocalization, and function in promoting the metastasis of cholangiocarcinoma and hepatocellular carcinoma

Successful construction of the eukaryotic expression plasmid pEGFP-C1-CsGRN. a Restriction enzyme identification of the recombinant plasmid pEGFP-C1-CsGRN. DNA ladder 5000 (Lane M), double enzyme digestion of pEGFP-C1-CsGRN (Lane 1), recombinant plasmid pEGFP-C1-CsGRN (Lane 2), empty vector pEGFP-C1 (Lane 3). b Sequencing data from recombinant plasmid pEGFP-C1-CsGRN and CsGRN gene were completely matched. (TIF 206 kb