Figure 7
From: Analysis of Wnt signalling dynamics during colon crypt development in 3D culture
Quantitative analyses of compartment β-cat and E-cad dynamics in colonospheres.
(A) Intensity projections of 3D β-cat (nuclear, cytosol or membrane compartments) and E-cad (membrane only) of day 2 colonospheres. (B) Relative subcellular compartment β-cat intensity of colonospheres under Wnt3A stimulation or CHX inhibition (1, 2, 3, 4.5 or 6 hours). Nuclear β-cat increased significantly after 2 hours of Wnt3A stimulation and decreased significantly after 2 hours of CHX inhibition. Relative cytosolic β-cat increased significantly after 2 hours of Wnt3A stimulation while under CHX inhibition, a slow decrease was observed which became significant after 4.5 hours of inhibition. Relative membrane β-cat increased slightly under Wnt3A stimulation (significant after 2 hours of stimulation) with a very slow decrease observed under CHX inhibition. (C) Average compartment E-cad intensity (all T0 to 6 hrs) for either Wnt3A or CHX treatment. The nuclear E-cad intensity detected was treated as background control (the antibodies used recognize only E-cad extracellular domain). In both treatments, the average E-cad intensity/pixel for both nuclear and cytosol compartments are similar, rendering cytosolic E-cad intensity detected as background. E-cad analyses therefore only consisted of the membrane compartment (minus the corresponding nuclear intensity as background correction). (D) The relative E-cad levels (background corrected) under Wnt3A stimulation or CHX inhibition. E-cad levels increased variably by 20% (p > 0.05 for each time-point) after 6 hours of Wnt3A stimulation. CHX decreased the E-cad levels by 20% after 3 hours (p < 0.05). (E) Compartment proportions (% nuclear, cytosol or membrane) of β-cat intensities were tabulated for all colonospheres under the two treatments (T0-T6 h). No change in compartment proportions was observed under either Wnt3A or CHX perturbations. Wnt3A n = 5, CHX n = 3, statistical test: single-factor anova analysis relative to T0: *p < 0.05, **p < 0.01, ***p < 0.001. Total colonospheres count:296. Error bars: ± SEM
