Figure 6: TRAF3IP1 mutations lead to epithelialization and polarity defects.

(a) mIMCD3 cells grown until confluence on filters were subjected to Ca²⁺-free medium to disrupt the tight junctions. Six hours after Ca²⁺ addition, cells were analysed by immunofluorescence using the apical marker Gp135 (red) and β-catenin (light blue) to stain the cell junctions. Scale bar, 10 μm. (b) Following Ca²⁺ switch, tight junction re-formation was assessed by measurement of trans-epithelial resistance (TER) at different time points (mean ± s.e.m. of n=5 independent experiments, two-way ANOVA; NS: not-significant, ***P<0.001 at 6 h). (c) Height of mIMCD3 cells grown on filters measured as the distance from the base to the top of the cells (GFP staining, not shown; mean ± s.d. of n≥20, from 3 independent experiments, ***P<0.001, Bonferonni's multiple-comparison test). (d) Expression of the apical marker Gp135 was analysed by Western blot with α-tubulin as a loading control. (e) mIMCD3 cells grown in matrigel 3D matrix to form spheroids were stained for ZO1 (tight junctions, red) and analysed by confocal microscopy. Arrows indicate ZO-1 at the apical junctions, while arrow heads point to mislocalized ZO-1. Equatorial sections of representative spheres are shown for each cell line. Scale bars, 10 μm. (f) Percentage of abnormal spheroids (no/small lumen filled with cells) (mean ± s.d., n=80 spheroids from 2 independent experiments, ***P≤0,001, **P<0.002, Bonferonni's multiple-comparison test).