A comprehensive model of WNT/,B-catenin signaling | We validated the presented model of WNT/fi-catenin signaling against independent in-silico and in-Vitro data [27, 49]. |
Abstract | Here we apply a combined in-vitro and in-silico approach to investigate the spatio-temporal regulation of WNT/[3-catenin signaling during the early neural differentiation process of human neural progenitors cells (hNPCs), which form a new prospect for replacement therapies in the context of neuro-degenerative diseases. |
Abstract | The model’s predictive ability is demonstrated under a wide range of varying conditions for in-vitro and in-silico reference data sets. |
Author Summary | Human neural progenitor cells offer the promising perspective of using in-vitro grown neural cell populations for replacement therapies in the context of neurodegenerative diseases, such as Parkinson’s or Huntington’s disease. |
Author Summary | Based on a combined in-vitro and in-silico approach we demonstrate an elaborate interplay between endogenous ROS and lipid raft dependent WNT/beta-catenin signaling controlling the nuclear beta-catenin levels throughout the initial phase of neural differentiation. |
Conclusion and Outlook | In a combined in-vitro and in-silico approach we find strong evidence, that cell fate commitment in human neural progenitor cells is driven by two distinct fi-catenin signaling mechanisms. |
Introduction | NPCs provide a new, promising basis for the in-vitro growth of neuron populations that can be used in replacement therapies for neurodegenerative diseases, such as Parkinson’s or Huntington’s diseases [6, 7]. |
Results/Discussion | We use literature values as often as possible and fit the remaining parameters to experimental measurements of nuclear fi-catenin dynamics during in-vitro differentiation of ReNcell VM 197 cells. |
Results/Discussion | To further test the calibrated/fitted model we apply cross-validation by reproducing existing in-silico and in-vitro data (measurements of fi-catenin concentration under different WNT stimuli). |
transcription signal. | Therefore we investigate the mutual influence of lipid rafts on WNT-signaling during the in-vitro differentiation of immortalized human neural progenitor cells (ReNcell VM197). |
transcription signal. | The model is based on experimental data as well as literature values and has been extensively validated against in-vitro and in-silico data under a Wide range of varying conditions. |
Statistical analysis | The mean and standard deviations of DMSO and Taxol controls for the in-vitro tubulin polymerization assays were calculated and used to scale the compound OD readout between different runs to normalize the heterogeneity of the reaction. |
Statistical analysis | All the statistical analysis for in-vitro tubulin polymerization assays was performed using Microsoft Excel. |
Supporting Information | (A) 212 antimitotic compounds clustered into 85 distinct chemical similarity sub-networks of which 23 clusters contained annotated anti-tubulin agents (green); additionally 54 novel tubulin-targeting chemotypes (yellow) were identified from in-vitro tubulin polymerization assays. |
Supporting Information | Of the 51 compounds predicted to be targeting microtubules, 36 compounds (71%) had more than 20% fold change in in-vitro tubulin polymerization assay and 14 had no measurable effect. |
Supporting Information | (B) Tubulin polymerization kinetics for 7 novel tubulin destabilizers (6—12), based on a phenyl-sulfanyl-thiazol-acetamide scaffold, using an in-vitro tubulin polymerization assay. |
Target validation of mitotic compounds from CSNAP predictions | Based on target prediction, we selected microtubules (0c and B-tubulin) as our target for in-vitro validation. |
Target validation of mitotic compounds from CSNAP predictions | To test CSNAP’s prediction that 51 of the 212 mitotic compounds were targeting microtubules, we reacquired all 212 compounds and tested their ability to perturb micro-tubule polymerization (stabilize or destabilize microtubules) in an in-vitro microtubule polymerization assay at 50uM concentration (Fig. |
Target validation of mitotic compounds from CSNAP predictions | In addition, in-vitro testing led to the discovery of 96 additional compounds for a total of 132 anti-tubulin agents, including structurally diverse compounds covering ~54 novel chemotypes not discovered in previous chemical screens (S3 Table). |