Fig. 5.

Treatment with the MNK1 inhibitor CGP 57380 reverses effects of the identified deactivating CDH1 variants in two cellular models. a Western blot analysis of active β-catenin (detected using a non-phospho-Ser45-specific antibody) in the nuclear fraction of heterozygous (WT/A817V) and homozygous (A817V/A817V) E-cadherin A817V knock-in and E-cadherin wildtype (WT/WT) HEK293T clones after 24 h incubation with CGP 57380 (75 µM) or DMSO control. b Densitometric evaluation of protein bands from (a) revealed a CGP 57380-dependent significant reduction of nuclear active β-catenin normalized to lamin A/C for the A817V/A817V clone, but not for the WT/A817V or WT/WT clones (mean ± SD of three independent experiments). c Western blot analysis of phospho-AKT (Ser473) and AKT (pan) in lysates of A817V knock-in and E-cadherin wildtype HEK293T clones after 24 h treatment with CGP 57380 (75 µM) or DMSO control. d Densitometric evaluation of the protein bands from (c) revealed a CGP 57380-dependent significant reduction of the ratio of phospho-AKT (Ser473) to AKT (pan) levels in WT/A817V and A817V/A817V cells, but not in WT/WT cells (mean ± SD of three independent experiments). e By MTS assay, cell viability after treatment with CGP 57380 (200 µM) for 72 h was significantly lower in WT/A817V and A817V/A817V cells than in WT/WT cells (mean ± SD of three independent experiments). f Detection of E-cadherin in CHO cells stably expressing E-cadherin WT, A592T or A817V after incubation with DMSO control or CGP 57380 by immunofluorescence. The reduced cell membrane localization of mutant E-cadherin observed after incubation with DMSO control could be reversed by treatment with CGP 57380 (75 µM) for 24 h; representative images of each cell line of one of three independent experiments are shown; scale bar 10 µm. Ctrl control; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns not significant (Student’s t test)