In prostate cancer cell line, LPA stimulates PC-3 cells motility through LPA1

mplexes, thus confirming the co-immunoprecipitation of both proteins and their interactions. In situ Hybridizations Demonstrated Integrin Transcription in Gonad Tissues A Novel RNAi Approach Revealed a Multinucleated Oocyte Phenotype for Smb-Int1 Deficiency For functional analyses we selected Smb-Int1 as target due to its unique role as universal dimerization partner of the schistosome a integrins, its interactions and co-localizations with the schistosome CTKs in gonad tissues. Additionally, we chose Sma-Int1 as target, since it showed similar localization as well as transcriptional regulation patterns. In analogy to previous inhibitor approaches we tried to inhibit Smb-Int1 function by Echistatin, a viper disintegrin representing the most potent inhibitor of b-integrins. Echistatin contains a cysteine-rich peptide containing the Arg-Gly-Asp sequence, which simulates a ligand-binding motif thus acting as potent, irreversible antagonist for b-integrin. Regarding cell culture experiments in mammalian cells Echistatin has an IC50 value of 0.130 nM. ML-128 Platyhelminth a/b-Integrin Receptors Adult S. mansoni couples were treated for 5 days with 100 nM, or for 3 days with 500 nM Echistatin in vitro. During these time periods no changes in physiology or behavior were observed, and no morphological abnormalities compared to control couples were detected using confocal laser scanning microscopy. 6 Platyhelminth a/b-Integrin Receptors 7 Platyhelminth a/b-Integrin Receptors of Sma-Int1 were also detected in the ovary, the ootype-surrounding area, and the vitellarium of the female, the testes of the male, and the parenchyma of both genders. Transcripts of Sma-Int2 were exclusively detected in the ootype-surrounding area anterior the ovary. Antisense and sense transcripts of Sma-Int3 and Sma-Int4 were only detected in the ovary. No signals were detected using sense transcripts of Smb-Int1, Sma-Int1, and Sma-Int2. dp: dorsal part; vp: ventral part; vs: ventral sucker; scale bars as indicated. doi:10.1371/journal.pone.0052519.g004 As an additional possibility to affect integrin function we performed RNAi experiments according to recently established protocols. To this end schistosome couples were electroporated followed by soaking with dsRNAs specific for Sma-Int1 or Smb-Int1. After 5 days, only slight reductions of the corresponding transcripts of 1525% or 1020% were achieved compared to control worms electroporated without dsRNA. Morphological analyses by CLSM revealed no differences 19778726 between dsRNA-treated versus control worms. As an alternative we tested siRNAs as templates for the RNAi machinery in schistosomes. The siRNA sequences were predicted by a specific algorithm to find optimized target sequences, and a special sequence motif was added to the selected sequences increasing the efficiency of gene silencing. Four different siRNAs were designed for each of the two schistosome integrins. Attempts with individual siRNAs failed 19535597 to significantly reduce the amount of transcripts of Sma-Int1 or Smb-Int1. To overcome this limitation we combined all four siRNAs specific for Smb-Int1 using 2.5 mg of each siRNA. This combinatory approach led to a reduction of the Smb-Int1 transcript level down to 58% compared to transcript levels in control worms electroporated without siRNAs or Sma-Int1specific siRNAs. A similar combinatorial approach failed to reduce the amount of Sma-Int1 transcripts. CLSM analyses of the couples electroporated with all four Smb-Int1siRNA