The H520 cell line lacks adherens junctions and expresses EMT traits.

(A) E-cadherin immunofluorescence showing membrane-associated E-cadherin localization (arrows) in H1666 and H1650 cells. In contrast, E-cadherin expression is notably decreased in H520 cells in a diffuse cytoplasmic staining pattern. E-cadherin expression is undetectable in SCLC H60 cells. In all preparations nuclei were counterstained with DAPI (blue). (B) Representative immunoblots showing decreased E-cadherin, increased N-cadherin and vimentin, decreased integrin α5 expression and increased FAK phosphorylation on Tyr397 in H520 cells relative to H1666 cells. Immunoblot using an anti α/β tubulin antibody (bottom panel) shows equal loading of the gel. (C) EMT marker expression was also measured at the mRNA level by qRT-PCR. While levels of the E-cadherin transcript are elevated in H1666 relative to H520 cells, the levels of N-cadherin and vimentin transcripts are elevated in H520 cells relative to H1666 cells, in all three instances in a statistically significant manner. Error bars are standard error of the mean, and P-values are shown on top of each graph. (D) Immunoblot replicas were quantified by densitometric analyses of band intensity. Bars represent band intensity for each protein, normalized against the α/βtubulin loading control, and error bars are standard error of the mean. P values, shown on top of each graph, indicate that all the observed changes in protein expression were statistically significant. Statistical tests were assessed by Mann-Whitney tests when comparing two groups and Kruskal Wallis when comparing three groups or more. Statistical significance was defined as, *p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001.