nt-CM was potently blocked using the Wnt LRP5/6 co-receptor blocker Dkk1; this can be expected as signalling activation by Wnt protein requires LRP co-activation. However, in our experiments Dkk1 had no effect on the blocking of reporter activity when cells were activated through the oscillating Fz-MNP. This suggests that Fz-MNP are signalling through an LRP independent mechanism. This result is in agreement with the Western blotting data which showed no noticeable increase in LRP5/6 phosphorylation after treatment with FzMNP which would indicate receptor activation. This again suggests an alternative mode of Wnt/-catenin activation by Fz-MNP which warrants further investigation. At 24h Fz-MNP 13 / 18 Remote Activation of Wnt Signalling treated groups remained elevated. This is further evidence of a level of mechanoactivation of Wnt signalling and demonstrates that Fz-MNP are capable of causing OPC 8212 sustained Wnt pathway activation. Treatment with Wnt-CM also significantly activated reporter activity to comparable levels as Fz-MNP. Taken together, this would suggest that a lag phase or threshold exists after stimulation with Wnt-CM that must be overcome before Wnt signalling activity peaks. This observation is in agreement with work from Carthy et al who showed activation of a Wnt TCF reporter after 24h treatment with recombinant Wnt 3A. Although Dkk1 was unable to block Wnt pathway activation by Fz-MNP, reporter activation by both Fz-MNP and Wnt-CM was successfully blocked using a downstream Wnt signalling blocker- iCRT-14, which acts by disrupting -catenin’s association and interaction with TCF transcription factors which is required for the expression of Wnt target genes. Control particles IgG-MNP and RGD-MNP were used to assess the effects of generic membrane stimulation on pathway activation. In our experiments both control particles were found to have no activating effects on reporter activity over both time-points studied. Finally, gene expression analysis was performed over early-time-points to investigate generic mechano-stimulation of stress response genes which have previously been shown to respond to mechanical stimulation. C-Myc is an oncogene associated with stress response; it has also been identified as a Wnt responsive gene and expression of c-Myc has been shown to increase in response to Wnt treatment. Low levels PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19761586 of up-regulation in all groups are observed after one hour e.g. Fz-MNP treatment and Wnt 3A treatment all up-regulate c-Myc expression after 1h to similar levels. This is comparable to results from Gujral et al who showed c-Myc expression increases in the first 3h after Wnt stimulation in HEK293 cells. Furthermore, in this experiment the expression profile of Fz-MNP matches the expression profile produced by Wnt 3a closely, whereas treatment with control particles targeted to the Trek-1 ion channel produced a broadly different expression profile to Wnt3A and Fz-MNP with Magnet. This is an indication that the FzMNP and Wnt 3A are having a similar effect on c-Myc gene expression. COX2 expression has been shown to increase in response 
to cytotoxic stress e.g. cytokines, endotoxins, -radiation . Recent evidence PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19763758 has also shown that the COX-2 promoter harbours TCF/LEF response elements and that activation of canonical Wnt signalling by lithium or Wnt 3A results in increased COX2 mRNA expression. Our results again show low levels of expression elevation at 1 hour in all experimental groups without a clear pattern emerging