Journal: PLoS ONE

Article Title: Association of Nuclear-Localized Nemo-Like Kinase with Heat-Shock Protein 27 Inhibits Apoptosis in Human Breast Cancer Cells

doi: 10.1371/journal.pone.0096506

Figure Lengend Snippet: (A) The levels of NLK in total cell lysates of MCF10A, MCF7, and MDA231 (upper panel). Densitometric analyses of the total levels of NLK in MCF7, and MDA231 cell lines (Lower panel) relative to NLK protein expression levels in MCF10A cells as percentage (mean ± s.e.m., p<0.05, n = 6). Densitometric analysis of NLK immunoblots was performed in the linear range of detection, and absolute values were then normalized to actin. (B) Isolation of nuclear (left panel) and cytosolic (right panel) fractions by a subcellular fractionation assay followed by a Western blot analysis, using NLK, tubulin or lamin B antibodies. (C) Cellular distribution of NLK in MCF10A, MCF7 and MDA231 cells. Cells were grown on glass coverslips in 6-well plates. 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. Right panel shows percentage of cells harboring NLK localization in the nuclei, cytosol, or distributed equally between the nucleus and the cytosol. Six to eight microscopic fields of four independent staining were analyzed. (D) Immunohistochemical analyses of subcellular localization of NLK in two normal human breast tissues and in two invasive breast cancer tissues. (E) MCF7 cells were transfected with 40 nM control or NLK siRNA for 24 hours and trypsinized before fixation with 4% formaldehyde for 30 minutes and staining with Meyers's haematoxylin for 5 min. Cells were subsequently centrifuged at 1400 rpm for 5 min and cell pellet were resuspended in 70% ethanol overnight. Cells pellets were dehydrated in graded ethanol series, embedded in paraffin and stained using NLK antibodies.

Article Snippet: For HSP27 or NLK knockdown, MCF7 cells were transfected once or two times with 50 nM HSP27-siRNA (Santa Cruz Biotechnology, Inc. [sc-29350]), HSP27-siRNA (Cell Signaling Technology, Inc. [SignalSilence #6356]), NLK shRNA Plasmid (Santa Cruz Biotechnology, Inc, [sc-36079-SH]), Control shRNA Plasmid (Santa Cruz Biotechnology, Inc, [sc-108060], or NLK-siRNA oligos (TAG Copenhagen, Denmark [NLK-Sense: GAA CCT CAG CTC TGT CCG ATT ; NLK-Antisense: TCG GAC AGA GCT GAG GTT CTT]) using Lipofectamine 2000 (Invitrogen) in accordance with the supplier's recommendations.

Techniques: Expressing, Western Blot, Isolation, Fractionation, Staining, Immunohistochemical staining, Transfection, Control

Journal: PLoS ONE

Article Title: Association of Nuclear-Localized Nemo-Like Kinase with Heat-Shock Protein 27 Inhibits Apoptosis in Human Breast Cancer Cells

doi: 10.1371/journal.pone.0096506

Figure Lengend Snippet: (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.

Article Snippet: For HSP27 or NLK knockdown, MCF7 cells were transfected once or two times with 50 nM HSP27-siRNA (Santa Cruz Biotechnology, Inc. [sc-29350]), HSP27-siRNA (Cell Signaling Technology, Inc. [SignalSilence #6356]), NLK shRNA Plasmid (Santa Cruz Biotechnology, Inc, [sc-36079-SH]), Control shRNA Plasmid (Santa Cruz Biotechnology, Inc, [sc-108060], or NLK-siRNA oligos (TAG Copenhagen, Denmark [NLK-Sense: GAA CCT CAG CTC TGT CCG ATT ; NLK-Antisense: TCG GAC AGA GCT GAG GTT CTT]) using Lipofectamine 2000 (Invitrogen) in accordance with the supplier's recommendations.

Techniques: Incubation, Immunofluorescence, Confocal Microscopy, Transfection, shRNA, Control, Western Blot, DNA Fragmentation Assay, Staining, Immunoprecipitation, Purification, Lysis, SDS Page

Journal: PLoS ONE

Article Title: Association of Nuclear-Localized Nemo-Like Kinase with Heat-Shock Protein 27 Inhibits Apoptosis in Human Breast Cancer Cells

doi: 10.1371/journal.pone.0096506

Figure Lengend Snippet: (A) MCF7 cells were mock-treated, or transfected with two different siRNAs targeting HSP27 (siRNA1 HSP27 or siRNA2 HSP27) or with a control oligonucleotide, twice for 24 hours, followed by a Western blot analysis, using HSP27 and Actin. (B) MCF7 cells were transfected with siRNAs targeting HSP27 or with a control oligonucleotide, twice for 24 hours. Nuclear (N) and cytosolic (C) fraction from total cell lysate was fractionated and followed by a Western blot analysis, using NLK, lamin B, or tubulin antibodies. (C) The cellular distribution of NLK in MCF7 cells transfected two times within 48 hours with siRNA oligos against HSP27. After fixation, cells were permeabilized with 0.25% Triton X-100 solution, after which they were blocked with 1% BSA, and subsequently probed with antibodies against NLK (1:100) and HSP27 (1:100). After washing the coverslips, fluorescent antibodies: Alexa fluor 488 goat anti Rabbit (1:1000) or Alexa fluor 568 Donkey anti Goat (1:1000) were applied and nuclei were stained with DAPI. (D) MCF7 cells were transfected with siRNAs targeting NLK or with a control oligonucleotide, twice for 24 hours. Nuclear (N) and cytosolic (C) fraction from total cell lysate was fractionated and followed by a Western blot analysis, using NLK, HSP27, tubulin, or lamin B antibodies. (E) Endogenous levels of NLK and HSP27 in total cell lysates from MCF7 cells, mock-treated, or transfected with siRNA against HSP27 or with a control oligonucleotide, twice for 24 hours. (F) MCF7 cells, transfected with siRNA, were evaluated 24 hours after secondary transfections, using the NucleoCounter cell viability assay. Data (mean ± s.e.m., p<0.05, n = 3) are presented as the amount of viable cells in comparison with mock-transfected cells. (G) MCF7 cells, transfected with siRNA, were fixed and stained with propidium iodide, 24 hours after secondary transfections. Cells were visualized by fluorescence microscopy, and scored for apoptotic nuclear morphology. Data (mean ± s.e.m., p<0.05, n = 3) show the amount of apoptotic cells in comparison with mock-transfected cells. (H) The cellular distribution of NLK in MCF10A transfected with HSP27 for 24 hours using immunofluorescence staining and confocal microscopy (upper panel). Lower panel shows the levels of HSP27 in transfected MCF10A cells.

Article Snippet: For HSP27 or NLK knockdown, MCF7 cells were transfected once or two times with 50 nM HSP27-siRNA (Santa Cruz Biotechnology, Inc. [sc-29350]), HSP27-siRNA (Cell Signaling Technology, Inc. [SignalSilence #6356]), NLK shRNA Plasmid (Santa Cruz Biotechnology, Inc, [sc-36079-SH]), Control shRNA Plasmid (Santa Cruz Biotechnology, Inc, [sc-108060], or NLK-siRNA oligos (TAG Copenhagen, Denmark [NLK-Sense: GAA CCT CAG CTC TGT CCG ATT ; NLK-Antisense: TCG GAC AGA GCT GAG GTT CTT]) using Lipofectamine 2000 (Invitrogen) in accordance with the supplier's recommendations.

Techniques: Transfection, Control, Western Blot, Staining, Viability Assay, Comparison, Fluorescence, Microscopy, Immunofluorescence, Confocal Microscopy