Fig 1.
Schematic of current notion of the relationship between Wnt/β-catenin signaling, N-glycosylation, and intercellular adhesion.
Image adapted from [25].
Fig 2.
Processes are numbered 1–26. Reactions 1–10 represent steps of Wnt/β-catenin signaling involved in active β-catenin regulation in the absence of Wnt3a. Reactions 11–19 represent regulation of Wnt3a binding by both genetic regulation of DPAGT1 and N-glycosylation. Reactions 20–26 represent E-cadherin dynamics and AJ formation. Abbreviations: APC, adenomatous polyposis coli; β-cat, β-catenin; E-cad, E-cadherin; ER, endoplasmic reticulum; ERC, endocytic recycling compartment; GSK3, glycogen synthase kinase 3β; LRP, lipoprotein receptor-related proteins; M, membrane; TCF, T-cell factor. Image partly adapted from [41].
Fig 3.
(LEFT) Sensitivity to changes in individual reactions of fold-change (upon activation of Wnt/β-catenin signaling) in β-catenin, DPAGT1 mRNA and GPT, AJ, and E-cadherin adhesivity at steady-state. Reaction labels refer to numbering used in Fig 2; repeated numbers used for processes described by more than one parameter. Reaction labels along the horizontal axis are organized from left to right in order of decreasing sensitivity averaged over all molecules in network. (RIGHT) Dependence of fold-change in chosen molecules in time to changes in reaction 11 (i.e. binding equilibrium of β-catenin and TCF). The horizontal axis represents the factor by which the parameter describing binding dynamics is scaled.
Table 1.
Ranking of processes of RCN in terms of sensitivity of system to corresponding parameter change.
Fig 4.
Predicted values for a) adherens junctions (AJ) and b) adhesivity (σ) when numerically solving RCN model.
The four conditions simulated the experimental conditions: reference condition corresponds to physiological rate of β-catenin/TCF complex formation (i.e. equilibrium constant K11, S1 Table); dysregulated condition corresponds to ICG-001 treatment, modeled as a disruption in β-catenin/TCF complex formation (i.e. rate constant changed to 2×K11). Wnt “OFF” state is modeled with a total Wnt3a concentration WNT0 = 1 nM, and Wnt “ON” with WNT0 = 28.062 nM.
Fig 5.
MDCK cells were treated with either conditioned media with (WCM) or without (CCM) Wnt3a, and either no inhibitor (DMSO) or ICG-001.
a) Representative immunoblots (IBs) of ABC in total cell lysates (TCL). Images were taken from a single membrane. Quantified intensity values are averages; errors bars represent standard error of the mean (SEM) (N = 4). Full IB with duplicates can be found in S3 Fig (supplemental). b) IBs for α-catenin and E-cadherin in E-cadherin immunoprecipitates (IP). CCM TLC and WCM TLC represent input, with isotype controls (IP IgG) included. Blots were quantified and normalized to the ICG-001 condition in each activation state. * represents statistically significant difference, p<0.05.
Fig 6.
E-cadherin mobility shift from treatment of total cell lysates with glycosidases: Endoglycosidase H (EndoH) or Peptide-N-Glycosidase F (PNGaseF).
a) Immunoblots of E-cadherin from cells grown without exogenous Wnt3a (CCM). b) Immunoblots for lysate from cells grown in exogenous Wnt3a (WCM). Cells were grown in the presence of either no inhibitor (DMSO) or ICG-001.
Fig 7.
a) Average magnitude of velocity field from PIV analysis in MDCK cell sheets and b) lateral correlation length at migrating wound edge. The four conditions correspond to treatment with: Either conditioned media with (WCM) or without (CCM) Wnt3a, and either no inhibitor (DMSO) or ICG-001. Values are the average (errors bars represent SEM) of three independent experiments for 27 time points in each (N = 81). ** and * represent statistically significant difference, p<0.005 and p<0.05 respectively.
Fig 8.
Movement angle is the angle between expected sheet direction (i.e. wound closing or 0°) and PIV determined velocity vectors.
Color bar indicates fraction of all PIV vectors with a particular orientation. Reference condition is DMSO treated and dysregulated condition is ICG-001 treated. CCM stands for control conditioned media; WCM stands for Wnt3a conditioned media.