Mutant 4 restored the E-cadherin to cell membrane and suppressed cell invasiveness in H460 cells.

<p>H460 cells were transiently transfected with GFP-siRNA-p120ctn plasmids. 24 hours after transfection, an aliquot of the cells was transfected again with one of the five p120ctn isoform 1A deletion mutants M1–5 cDNA plasmids or with empty vector as control (The group transfected with vector alone was not included in the data). (<b>A</b>) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal indicates expression of GFP from GFP-siRNA-P120ctn construct in image I and represents combined expression of GFP from GFP-siRNA-P120ctn and M1–5-GFP in images III, V, VII, IX and XI. GFP from GFP-si-P120ctn, M1–M3, and M5-GFP was expressed in the nucleus and cytoplasm. GFP from GFP-si-P120ctn and M4-GFP was expressed in the nucleus, cytoplasm and on the cell membrane (IX). Expression or repletion of M4 mutant (IX) restored E-cadherin on the cell membrane (X), while expression of the other mutants had no significant effects on E-cadherin levels. (<b>B</b>) The levels of E-cadherin were then analyzed by Western blot assay. Expression of M1–M5 mutants were detected by using antibody against GFP. The results showed that expression of M4 mutant up-regulated the E-cadherin levels, whereas the other mutants had no effects on the E-cadherin levels. (<b>C</b>) The invasiveness of H460 cells was analyzed by Matrigel invasion assay. Repletion of M4 mutant reduced the cell invasiveness in comparison with the group of p120ctn ablation (Si-p120ctn) (**, <i>P</i><0.01), while repletion of M2 mutant enhanced the invasiveness (*, <i>P</i><0.01). Repletion of the other mutants (M1, M3 and M5) did not show significant effects on cell invasiveness in comparison with the group of 120ctn ablation.