Caspase-3 activity increases following QLT-0276 treatment in paired females.

<p>Result of the caspase-3 activity assay showing that compared to a DMSO-treated control (left) the activity of caspase-3 of <i>S</i>. <i>man</i>soni increased significantly (p = 0.01) in paired females treated with 100 μM QLT-0276 (right) as determined spectrophotometrically (ΔOD₄₀₅).</p

SmShb is a partner of the JNK pathway.

<p>cRNA (40 ng) of SmVKR1(A) or SmVKR2 F₉₄₉Y (B) were injected in oocytes without or with cRNA (25 ng) encoding SH2_SmShb or SH2-deleted SmShb (SmShb^ΔSH2). After two hours, a second injection of cRNA encoding SmShb (35 ng) was performed. Oocytes were further incubated for 3 hours then lysed. JNK phosphorylation was detected in oocyte lysates by western blotting using anti-phospho JNK antibodies. Pre-incubation of the SH2 domain with SmVKR1 and with SmVKR2 F₉₄₉Y blocked the capacity of SmShb to induce JNK pathway.</p

Egg production of worm couples decreases following QLT-0276 treatment <i>in vitro</i>.

<p>Analyses of egg production of adult <i>S</i>. <i>mansoni in vitro</i> following inhibitor treatment with the ILK inhibitor QLT-0276, which was used in different concentrations as indicated. Egg production significantly decreased from 48 h post treatment on in a concentration-dependent manner.</p

SmShb interacts with ligand-activated SmVKR1 and SmVKR2 receptors.

<p>(A) cRNA encoding V5-tagged SmVKR1 WT or SmVKR1 Y₉₇₉F were injected with or without cRNA encoding HA-tagged SmShb in <i>Xenopus</i> oocytes. Following their expression, full-length receptors were activated by L-Arg to induce their auto-phosphorylation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163283#pone.0163283.ref011" target="_blank">11</a>]. Proteins were then analysed by immunoprecipitation (IP) using anti-V5 or anti-HA antibodies followed by western blot (WB) using anti-V5, anti-HA or PY20 (anti-tyrosine phosphorylation) antibodies. Results demonstrated that only phosphorylated SmVKR1 WT bound to and phosphorylated SmShb as revealed by PY20 antibodies. (B) cRNA encoding V5-tagged SmVKR2 WT or SmVKR2 F₉₄₉Y were injected with or without cRNA encoding HA-tagged SmShb in <i>Xenopus</i> oocytes. Following their expression, full-length receptors were activated by Ca⁺⁺ to induce their auto-phosphorylation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163283#pone.0163283.ref011" target="_blank">11</a>]. Proteins were then analysed by immunoprecipiration (IP) using anti-V5 or anti-HA antibodies followed by western blot (WB) analysis using anti-V5, anti-HA or PY20 (anti-tyrosine phosphorylation) antibodies. Results indicated that phosphorylated SmVKR2 WT did not bind SmShb whereas the mutated V5-SmVKR2 F₉₄₉Y bound to and phosphorylated SmShb.</p

Results of germinal vesicle breakdown assays in <i>Xenopus</i> oocytes and subsequent coimmunoprecipitation experiments.

<p>Results of germinal vesicle breakdown assays in <i>Xenopus</i> oocytes and subsequent coimmunoprecipitation experiments.</p

The juxtamembrane tyrosine (Y979) of SmVKR1 is required for SmShb binding.

<p>cRNAs encoding the intracellular domains (ICD) of Myc- SmVKR1 YYRE or SmVKR1 YYRE Y₉₇₉F were co-injected in <i>Xenopus</i> oocytes with HA-SmShb and soluble extracts were immunoprecipitated (IP) by anti-Myc or anti-HA antibodies. Immune complexes were analyzed by Western Blot (WB) using anti-Myc or anti-HA antibodies. SmShb co-precipitated with SmVKR1 ICD YYRE but not with SmVKR1 ICD YYRE Y₉₇₉F.</p

SmVKR-SmShb interaction induces JNK pathway.

<p><i>Xenopus</i> oocytes were co-injected with cRNAs encoding V5-SmVKR1 WT or Y₉₇₉F or V5-SmVKR2 WT or F₉₄₉Y and SmShb and incubated with 1μM L-Arginine or 1mM Ca⁺⁺ respectively. Oocyte lysates were analyzed by Western blot to detect phosphorylation of ERK2, Akt, S6K and JNK as described in Materials and Methods. In the absence of SmShb, SmVKR1 WT and Y₉₇₉F activated ERK2, Akt, S6K and JNK pathways whereas SmVKR2 WT and F₉₄₉Y activated ERK2, Akt, S6K but not JNK. However, in the presence of SmShb, SmVKR1 WT and SmVKR2 F₉₄₉Y did no more activate ERK2, Akt, S6K. While JNK pathway was maintained with SmVKR1 WT, it was specifically induced by SmShb in the case of the SmVKR2 F₉₄₉Y mutated and able to bind SmShb. In both cases, GVBD dependent on ERK2 and Akt activation was inhibited. SmShb did not affect signaling by SmVKR1 Y₉₇₉F or SmVKR2 WT, which were not able to interact with SmShb.</p

Effects of SmShb knock-down by RNA interference in adult worms.

<p>Worm couples were electroporated and incubated for 7 days either with dsSmShb or irrelevant dsLuc (control) as described in Materials and Methods. (A) Levels of SmShb transcripts were determined by quantitative RT-PCR in each worm sample. Interference led to a reduction of about 80% in the level of SmShb transcripts compared to controls. Statistical analysis was performed using the Student’s t-test and values are expressed as mean ± SEM of four determinations (*** p<0.001). (B) CLSM images of whole-mount preparations of male (a, b) or female (c, d) worms issued from worm couples electroporated with dsLuc (a, c) or dsSmShb (b, d). Accumulation of sperm in testicular lobes is observed in dsSmShb-treated males (b). Less mature oocytes were present in the posterior part of the female ovary (d). (ov: ovary; mo: mature oocytes; io: immature oocytes; t: testes; sv: seminal vesicle; arrows indicate mature sperm; scale bar = 20μm)</p

Complex formation induces ERK2, JNK and AKT pathways in <i>Xenopus</i> oocytes in a ligand-independent way.

<p>Western blot analyses of signaling pathways triggered by Smβ-Int1 complex-mediated SmVKR1 activation in <i>Xenopus</i> oocytes. The schistosome complex members were expressed in <i>Xenopus</i> oocytes for 5 h with or without ligand. Oocyte lysates were analyzed to investigate the phosphorylation state of ERK2, JNK and AKT following SmVKR1- Smβ-Int1 complex formation. In the absence of L-Arg as ligand, and in case of the combination of Smβ-Int1, SmILK, SmPINCH, SmNck2, and SmVKR1 (lane 1) phosphorylation of ERK2 (ERK2P), JNK (JNKP), and AKT (AKTP) was observed. In cases of using mutated forms (SmILKΔAnk1, lane 2; SmPINCHΔLIM1, lane 3; SmPINCHΔLIM4, lane 4; SmNck2ΔSH3, laneΔ 5; SmVKR1 KO, lane 6) or the ILK inhibitor QLT-0267 (1 μM; lane 7), no phosphorylation of ERK2, JNK, and AKT was detected. As expected, phosphorylation of ERK2, JNK, and AKT was observed in SmVKR1-expressing oocytes stimulated by L-Arg (1 μM), which served as positive control (lane 8).</p

Localization of SmShb transcripts in sections of paired adult worms by <i>in situ</i> hybridization.

<p>SmShb transcripts were detected in mature oocytes (A), in testes (B) and lightly in the vitellarium (C). Sense probe of SmShb was used as control in D, E and F. (m: male; f: female; mo: mature oocytes; t: testes; v: vitellarium; scale bar = 20μm)</p