Cytosolic localization of NLK induces cell death.

<p>(A) MCF7 and MCF10A cells were transfected for 24 hours with vectors encoding FLAG-tag fusions of full-length NLK (WT), or catalytically inactive NLK mutants (K155M and T286V). NLK localization (Green) was visualized by immunofluorescence staining and confocal microscopy. The nuclei of the cells were counter-stained with DAPI (Blue). (B) MCF7 cells were transfected with vectors encoding FLAG-tag fusions of full-length NLK (WT) or catalytically inactive NLK mutants (K155M and T286V), and counted 24, 48, or 72 h post transfection, using a Countess Automated Cell Counter. Data (mean ± s.e.m., p<0.05, n = 4) are expressed as number of viable cells at each time point. (C) MCF7 cells were mock-treated or transfected for 24 hours with WT-, K155M-, or T286V-NLK. The cells were fixed and stained with propidium iodide in order to count the cells displaying an apoptotic nuclear morphology. Data (mean ± s.e.m., p<0.05, n = 3) show the percentage of apoptotic cells in comparison with cells of mock-treated controls. (D) MCF7 cells were mock-treated or transfected for 24 hours with WT-, K155M-, or T286V-NLK. After 48, or 72 hours cells were subjected to DNA fragmentation assay. Data (mean ± s.e.m., p<0.05, n = 3) represent the percent of sub G0 population. Right panels shows the levels of Flag tagged WT-, K155M-, or T286V-NLK overexpressed in MCF10A and MCF7 cells.