Visual model of the inhibitor effects on transcription of genes involved in eggshell-formation.

<p>The inhibition of Src kinases targeted by Herb A and TβRI by TRIKI revealed an influence of both inhibitors on the transcription of genes known to play roles in eggshell formation such as Smp14, Smp48, eggshell precursor protein, SmTYR1 and fs800. Compared to TRIKI treatment, however, a stronger effect on transcription was observed when adult schistosome couples were treated with Herb A. From this we conclude cooperative signal- transduction activities during eggshell-formation with a more dominant role of Src kinases in promoting the expression of involved genes.</p

Comparison of qPCR and microarray results of genes involved in eggshell-formation processes.

<p>Summary of the log₂ratio (treated/control) obtained by qPCR and microarray analyses of genes coding for proteins proven or hypothesised to be involved eggshell-formation processes following treatment of paired female schistosomes with either 300 nM TRIKI, 4.5 µM Herb A, or the combination of both inhibitors. The investigated genes were the eggshell precursor proteins Smp14 and Smp48 as well as a predicted eggshell precursor protein (precursor), the tyrosinase 1 (SmTYR1), and a gene similar to fs800. For each method three biological replicas were used, each with two technical replicas for the microarray analysis (except microarray 2) and three technical replicas for the qPCR. The mean of the technical replicas was calculated and is presented with standard deviations.</p

<i>In situ</i> hybridization localizing the schistosome homologs of hippocalcin, ORAI-1, a predicted egg-shell precursor protein gene, and calmodulin-4.

<p>Sections (5 µm) of adult schistosome couples (males and females are indicated), were hybridized with DIG-labeled antisense-RNA probes of hippocalcin (A, B), ORAI-1 (D, E), eggshell precursor protein gene (G, H), and calmodulin-4 (J, K). For control, DIG-labeled sense-RNAs probes of hippocalcin (C), ORAI-1 (F), eggshell precursor protein gene (I), and calmodulin-4 (L) were used. Signals were observed in the ovary (o), the vitellarium (v), around the ootype (ot) where Mehli's gland is located, and the testes (t). Scale bar: 20 µm.</p

Comparison of qPCR and microarray results following Herbimycin A-treatment.

<p>Transcriptional changes from the Herb A-treatment of adult schistosome females detected in qPCR and microarray data were calculated as log₂ratios (treated/control). According to the microarray analysis SmActRIIb, calmodulin-4, and hsp70 showed enhanced transcription (A), whereas tetraspanin-1, SmSmad 4, and Smp48 were detected as transcriptionally repressed (B). For each method three biological replicas were used, each with two technical replicas for the microarray analysis (except microarray 2) and three technical replicas for the qPCR. The mean of the technical replicas was calculated and is presented with standard deviations.</p

Comparison of qPCR and microarray results following the combined inhibitor treatment.

<p>Transcriptional changes from the simultaneous TRIKI- and Herb A-treatment of adult schistosome females detected in qPCR and microarray data were calculated as log₂ratios (treated/control). The investigated genes with enhanced transcription, as determined by microarray analysis, were calmodulin-4 and hsp70 (A), the genes with repressed transcription comprised a gene similar to fs800, an eggshell precursor protein, tetraspanin 1, SmTYR1, and cathepsin S (B). For each method three biological replicas were used, each with two technical replicas for the microarray analysis (except microarray 2) and three technical replicas for the qPCR. The mean of the technical replicas was calculated and is presented with standard deviations.</p

Venn diagram of differentially transcribed genes identified by different inhibitor treatments.

<p>Numbers of differentially transcribed protein-coding genes of all three microarray experiments (“TRIKI”, “Herbimycin A”, “Combined treatment”). Each circle represents the microarray result of one of the inhibitor treatments. The intersections show the numbers of corresponding genes differentially transcribed following treatment with different inhibitors.</p

Hierarchical clustering of differentially transcribed genes (q≤0.03) following TRIKI-treatment, Herbimycin A-treatment, and combined inhibitor treatment.

<p>Hierarchical clustering of (A) 2330 (TRIKI), (B) 1021 (Herbimycin A), and (C) 411 (combined inhibitors) differentially transcribed genes (q≤0.03) of treated and control female schistosomes. Columns represent three biological replicas as well as two technical replicas (A, TRIKI: columns TRIKI/DMSO 6 and 5, as well as TRIKI/DMSO 1 and 2 represent technical replicas of the first two biological replicas, respectively; TRIKI/DMSO 3 represents the 3^rd biological replicate. B, Herbimycin A: columns Herb/DMSO 2 and 1, as well as Herb/DMSO 5 and 6 represent technical replicas of the first two biological replicas, respectively; Herb/DMSO 3 the 3^rd biological replicate. C, combined inhibitors: columns H+T/DMSO 6 and 5, as well as H+T/DMSO 4 and 3 represent technical replicas of the first two biological replicas, respectively; H+T/DMSO 2 represents the 3^rd biological replicate). Each line shows the calculated log₂ratio (treated/control) of the transcription of a gene; genes with a repressed transcription were colored in green (A, TRIKI: 565 genes; B, Herbimycin A: 302 genes; C, combined inhibitors: 157 genes) and genes with an enhanced transcription in red (A, TRIKI: 1765 genes; B, Herbimycin A: 719 genes; C, combined inhibitors: 254 genes).</p

Comparison of qPCR and microarray results following TRIKI-treatment.

<p>Transcriptional changes from the TRIKI-treatment of adult schistosome females detected in qPCR and microarray data were calculated as log₂ratios (treated/control). The investigated genes were SmTβRI, SmActRIIb, a protein similar to fs800, eggshell precursor, and Na/K-pump as representative for enhanced transcription within the microarray data set (A). The selected genes representing the set of genes with repressed transcription within the microarray data set (B) were calmodulin-4, tetraspanin 18, SmSmad 4, and snurp. For each method three biological replicas were used, each with two technical replicas for the microarray analysis (except microarray 2) and three technical replicas for the qPCR. The mean of the technical replicas was calculated and is presented with standard deviations.</p

GVBD assays confirming SmAbl2 inhibition by Imatinib.

<p>Results of the GVBD assays performed in <i>Xenopu</i>s oocytes to analyze schistosome-kinase activities under inhibitor influence. Herbimycin A completely inhibited SmTK3 TK enzymatic activity (circles, black) even at 0.01 µM, whereas 10 µM was needed to stop inhibit SmTK6 (squares, grey)-induced GVBD <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002923#pntd.0002923-Beckmann3" target="_blank">[38]</a>. At this concentration the GVDB-inducing TK activities of SmAbl1 (rhombus, grey) and SmAbl2 (triangle, black) were reduced by only 40% or 10%, respectively. Imatinib completely inhibited GVBD induced by the TK activities of SmAbl1 (at 1 µM) or SmAbl2 (at 0.1 µM), whereas SmTK6-induced GVBD was stopped at 100 µM, a concentration that reduced SmTK3 TK-induced GVBD by only 10% <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002923#pntd.0002923-Beckmann3" target="_blank">[38]</a>.</p

Alignment of the TK domains of human and schistosome Abl kinases.

<p>Alignment: Structural alignment of the binding sites of human (HsAbl1, HsAbl2) and <i>S. mansoni</i> (SmAbl1, SmAbl2) Abl kinases focused on the highly conserved catalytic tyrosine domain <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002923#pntd.0002923-Vanderstraete1" target="_blank">[37]</a>. Amino acid residues partnering in directional interactions with Imatinib are highlighted ocher and red for SmAbl1/2 and HsAbl1/2, respectively. Amino acids D568 and K457 of SmAbl1 are colored light teal, because they are not direct interaction partners to Imatinib but utilize a water molecule.</p