TNF-α and etoposide promotes accumulation of NLK in the cytoplasm.

<p>(A) MCF7 breast cancer cells were incubated in the absence or presence of 50 ng TNF-α for 18 hours. The localization of NLK was examined with immunofluorescence followed by confocal microscopy. (B) MCF10A cells were transfected with mock, shRNA targeting NLK, or with a control ShRNA for 24 hours, followed by a Western blot analysis of the total cell lysate, using NLK and Actin (left panel) or subjected to DNA fragmentation assay (right panel, n = 2). Data represent the percent of sub G0 population. (C) MCF7 cells were transfected with shRNA targeting NLK, or with a control ShRNA for 24 hours and treated with etoposide (upper panel, 10 µM for 16 hours) or TNF-α (lower panel, 50 ng TNF-α for 18 hours), followed by DNA fragmentation assay (n = 4). Data represent the percent of sub G0 population. (D) MCF10A cells were grown on glass coverslips in 6-well plates in triplicates. On the following day the cells were treated with DMSO or 100mM of etoposide for 12 hours. After fixation, cells were permeabilized with a 0.25% Triton X-100 solution. Cells were further blocked with 1% BSA, and probed with antibodies against NLK. Nuclei were counter-stained with DAPI. (E) MCF10A, MCF7 and MDA-231 cells were lysed and immunoprecipitated with anti-NLK antibody. Immunoprecipitates were purified by washing two times with lysis buffer containing high salt (0.625M NaCl), three times with PBS containing high salt (0.625 M NaCl), and two times with PBS. The pellet were incubated with 0.5 µg LEF1 and 1 mM ATP in 40 µl of kinase buffer (10 mM MgCl2, 10 mM HEPES (pH 7.4), and 1 mM DTT) for 30 minutes at 30°C. Samples were resolved by SDS-PAGE followed by a Western blot analysis, using phospho-LEF1 and total-LEF1 antibodies.