Fig. 3.

Generation and characterization of a HEK293T knock-in cell model harboring the CDH1:c.2450C > T p.(A817V) variant demonstrates its pathogenicity and affected pathomechanisms. a Schematic overview of the knock-in strategy using the CRISPR/Cas9 technology. The HDR template harboring the CDH1 variant also contained a synonymous PAM site variant. b Electropherograms of the target region in CDH1 exon 16 of the selected single cell clones showing two wildtype alleles (WT/WT), a heterozygous (WT/A817V) or homozygous (A817V/A817V) knock-in. The genotype of the A817V variant correlated with that of the synonymous PAM site variant. c The genotypes of the selected cell clones were confirmed by gel electrophoresis after TauI restriction digest of a PCR amplicon from the target region in CDH1 exon 16. d Using a slow aggregation assay, fewer cell–cell aggregates were observed in WT/A817V and A817V/A817V versus WT/WT cells after 72 h; representative images of one of three independent experiments performed in triplicate are shown; scale bar 200 µm. e, f In a wound healing assay analyzed 24 h after applying scratches, knock-in cells showed increased migration compared to WT/WT cells (e); relative cell migration (mean ± SD) was determined in four independent experiments performed in triplicate (f); scale bar 200 µm. g Using an MTS assay, no difference in cell viability was detected in knock-in compared to WT/WT cells after 44 h (mean ± SD of three independent experiments performed in triplicate). h, i By immunoprecipitation (IP) of β-catenin (h), a significant decrease of bound E-cadherin was detected in A817V/A817V compared to WT/WT cell lysates (mean ± SD of four independent experiments) (i) indicating impaired β-catenin binding of mutant E-cadherin. j By densitometric quantification of Western blot analyses (h), no differences in E-cadherin levels were observed in knock-in versus WT/WT cell lysates (mean ± SD of four independent experiments). k By densitometric quantification of Western blot analyses (h), total β-catenin levels were significantly increased in A817V/A817V versus WT/WT cell lysates (mean ± SD of four independent experiments). l, m Western blot analyses (l) and densitometric quantification (m) of the cytosolic and nuclear fractions showed a significant increase of active β-catenin levels in both fractions of A817V/A817V compared to WT/WT cells (mean ± SD of two independent experiments). WT wildtype; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns not significant (Student’s t test)