Fig. 2.

Colorectal cancer cells with EPHB1 receptor mutations R90C, R170W, R351L and D762N had reduced compartmentalisation in presence of EFNB1. A Upper panel, positions of the mutants in the full length EPHB1 protein. Lower panel, in vitro compartmentalisation assays by co-culturing DLD-1 cells expressing eGFP along with either wild-type EPHB1 or R90C, R170W, R351L and D762N mutants with DLD-1 cells expressing mCherry with or without EFNB1 ligand. Representative confocal images from 10 randomly chosen image fields for each type. Arrows, examples of large (> 50 cells), homogeneous GFP⁺ cell clusters indicative of cell sorting and compartmentalisation. B Quantitative results from the compartmentalisation experiments. Cell distribution was quantified by counting the percentage of GFP⁺ cells forming clusters of different sizes. In co-cultures of EFNB1 ligand with EPHB1 and its four mutated versions R90C, R170W, R351L and D762N, significantly lower percentage of GFP.⁺ cells were distributed into large homogeneous clusters (> 50) as compared to the Wt. Mann–Whitney U test was used to calculate large cluster (> 50 cells) difference between each mutant with or without EFNB1 against the respective positive control condition with EPHB1 wild-type. This experiment was performed at least twice and imaged with five random fields from each experiment. Here, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001