Journal: PLoS ONE

Article Title: P120-Catenin Isoforms 1 and 3 Regulate Proliferation and Cell Cycle of Lung Cancer Cells via β-Catenin and Kaiso Respectively

doi: 10.1371/journal.pone.0030303

Figure Lengend Snippet: ( A ) Western blot analyses show the increased expression of Kaiso in A549 cells transfected with Kaiso cDNA. Kaiso overexpression remarkably down-regulated the expression of cyclin D1 (p = 0.000) and cyclin E (p = 0.003). ( B ) Western blot analyses show reduced expression of Kaiso in SPC cells transfected with Kaiso siRNA. Kaiso interference significantly increased the expression of cyclin D1 (p = 0.001) and cyclin E (p = 0.012). Overall findings suggest that Kaiso could regulate the expression of cyclin D1 and cyclin E. ( C ) Chromatin immunoprecipitation (ChIP) assay confirmed the Kaiso monoclonal antibody could precipitate the cyclin D1 gene promoter fragment containing KBS. Kaiso could bind to KBS of cyclin D1 promoter. All the comparisons are made to the group of cells transfected with vector alone.

Article Snippet: Lysates were quantified by Bradford assay and equal amounts of total protein were used for immunoprecipitation with the anti-p120ctn, anti-p120ctn-1, 2 (6H11, Upstate Biotechnology, LakePlacid, NY, USA), anti-GFP (Santa Cruz Biotechnology Inc, CA, USA) or anti-kaiso mAb.

Techniques: Western Blot, Expressing, Transfection, Over Expression, Chromatin Immunoprecipitation, Plasmid Preparation

Journal: PLoS ONE

Article Title: P120-Catenin Isoforms 1 and 3 Regulate Proliferation and Cell Cycle of Lung Cancer Cells via β-Catenin and Kaiso Respectively

doi: 10.1371/journal.pone.0030303

Figure Lengend Snippet: ( A and B ) Co-immunoprecipitation assays showed that p120ctn monoclonal antibody (the lower panel of left column in ), but not specific p120ctn-1,2 monoclonal antibody (6H11) (the lower panel of right column in ), can effectively precipitate Kaiso protein both in the nucleus and cytoplasm . However, Kaiso monoclonal antibody cannot effectively precipitate p120ctn (the upper panel of middle column in ). ( C ) Co-immunoprecipitation with sufficient p120ctn mAb after overexpression of p120ctn-1 or 3 isoform showed that overexpression of p120ctn-3 led to more Kaiso protein precipitated in A549 cells. No change was detected when p120ctn isoform 1 was overexpressed, compared with the control. Specific p120ctn-1, 2 monoclonal antibodies (6H11) could not precipitate Kaiso when p120ctn isoform 1 was significantly increased. Note the presence of p120ctn after coprecipitation with 6H11 but absence of kaiso in the precipitate, suggesting there is no significant interaction between p120 isoform 1, 2 and kaiso. Since there are mainly p120ctn isoforms 1 and 3 in both of the lung cancer cell lines, we thought that Kaiso might bind to p120ctn isoform 3 but not to p120ctn isoform 1 in vivo.

Article Snippet: Lysates were quantified by Bradford assay and equal amounts of total protein were used for immunoprecipitation with the anti-p120ctn, anti-p120ctn-1, 2 (6H11, Upstate Biotechnology, LakePlacid, NY, USA), anti-GFP (Santa Cruz Biotechnology Inc, CA, USA) or anti-kaiso mAb.

Techniques: Immunoprecipitation, Over Expression, In Vivo

Journal: Small GTPases

Article Title: RhoH participates in a multi-protein complex with the zinc finger protein kaiso that regulates both cytoskeletal structures and chemokine-induced T cells

doi: 10.1080/21541248.2016.1220780

Figure Lengend Snippet: RhoH and Kaiso interact and co-localize in Jurkat T cells. (A) Interaction of HA-RhoH and the Kaiso complex were confirmed by co-immunoprecipitation. Jurkat cells transduced with retroviral vectors expressing HA-RhoH-IRES-YFP were subjected to IP with anti-HA monoclonal antibody and analyzed by immunoblotting with anti-Kaiso monoclonal antibody. Position of interacting Kaiso is indicated by arrowhead. IgG control is shown in left lane. Size marker is shown on right. A representative immunoblot of 3 independent experiments that gave similar results is shown. (B) Co-localization of RhoH and Kaiso in Jurkat cells. Jurkat cells were stained with polyclonal anti-RhoH antibody (green), monoclonal anti-Kaiso antibody (red) and nuclear staining with DAPI (blue). Arrowheads indicate co-localization readily apparent in cytosol and areas of cell protrusion. Cell Images were captured by Zeiss LSM 700 Laser Scanning Confocal-Microscope with 63X magnification and processed by ZEN software (Zeiss). Panels on the right show higher magnification (126X) of cell outlined by box in lower magnification veiw. This result represents 3 independent experiments. Mouse IgG and rabbit IgG were used as negative controls respectively and showed no signals (data not shown).

Article Snippet: Finally, Kaiso or p120 catenin were detected with anti-Kaiso monoclonal antibody (Abcam, Cambridge, MA 1:500 dilution) or anti-p120 catenin antibody (BD Biosciences Transduction Laboratories, San Diego, CA) and HRP-coupled anti-mouse IgG antibody (1:2000, Cell Signaling Technology, Danvers, MA) using chemiluminescence (Cell Signaling Technology, Danvers, MA).

Techniques: Immunoprecipitation, Transduction, Expressing, Western Blot, Marker, Staining, Microscopy, Software

Journal: Small GTPases

Article Title: RhoH participates in a multi-protein complex with the zinc finger protein kaiso that regulates both cytoskeletal structures and chemokine-induced T cells

doi: 10.1080/21541248.2016.1220780

Figure Lengend Snippet: SDF-induced Kaiso co-localization in cell protrusions is RhoH dependent. (A) Co-localization of RhoH and Kaiso at cell protrusion sites and nucleus after SDF-1 stimulation. Jurkat cells were stimulated with SDF-1 (200 ng/ml) for the indicated time points, fixed and stained with polyclonal anti-RhoH antibody (green), monoclonal anti-Kaiso antibody (red) and nuclear staining with DAPI (blue). Cell images were captured by Zeiss LSM 700 Laser Scanning Confocal-Microscope with 63X magnification and processed by ZEN software (Zeiss). RhoH and Kaiso co-localized at cell protrusion sites 30 sec. after SDF-1 stimulation (indicated by arrow) then started to co-localize in the cytosol and nucleus by 2 min. This shift in localization increased over 15 min (indicated by arrowhead) with a concomitant reduction in membrane localization. The higher magnification images of 15 minutes stimulation are shown at bottom column. Represenative images of 3 independent experiments are shown. (B) Cell fractionation and immunoprecipitation of HA-RhoH and Kaiso complex after SDF-1 stimulation. Jurkat cells transduced with a retrovirus expressing HA-RhoH-IRES-YFP were stimulated with SDF-1 (200 ng/ml) for the indicated time points. Subsequently cells were separated into nuclear, cytosolic fractions. The cell lysates from each fraction were subjected to IP with anti-HA monoclonal antibody and analyzed by immunoblotting with anti-Kaiso monoclonal antibody. HA-RhoH transduced Jurkat cells were stimulated with SDF-1 (200ng/ml) for the times indicated and fractionated into nuclear and cytosolic fractions. The HA-immunoprecipitated fractions (20ug) were analyzed for purity by immune blotting with anti-GAPDH antibody (cytosol) and anti-Histone polyclonal antibody (nuclear). A representative example of 3 independent experiments is shown. Abbreviations: c, unstimulated control. (C) RhoH knockdown is associated with the loss of Kaiso localization at SDF-1-induced cell protrusion sites. Jurkat cells transduced with scrambled control or RhoH shRNA were stimulated with SDF-1 (200 ng/ml) for 2 minutes. Stimulated cells were stained with monoclonal anti-Kaiso antibody (purple), rhodamine-phalloidin (red) to detect F-actin and nuclear DAPI staining (blue). Cell images were captured by Zeiss LSM 700 Laser Scanning Confocal-Microscope with 63X magnification and processed by ZEN software (Zeiss). Note decreased intensity of cortical Kaiso staining and increase in F-action at cell protrusion sites (indicated by arrowheads) following SDF-stimulation in RhoH knockdown cells.

Article Snippet: Finally, Kaiso or p120 catenin were detected with anti-Kaiso monoclonal antibody (Abcam, Cambridge, MA 1:500 dilution) or anti-p120 catenin antibody (BD Biosciences Transduction Laboratories, San Diego, CA) and HRP-coupled anti-mouse IgG antibody (1:2000, Cell Signaling Technology, Danvers, MA) using chemiluminescence (Cell Signaling Technology, Danvers, MA).

Techniques: Staining, Microscopy, Software, Cell Fractionation, Immunoprecipitation, Transduction, Expressing, Western Blot, shRNA

Journal: Small GTPases

Article Title: RhoH participates in a multi-protein complex with the zinc finger protein kaiso that regulates both cytoskeletal structures and chemokine-induced T cells

doi: 10.1080/21541248.2016.1220780

Figure Lengend Snippet: Sequential SDF-1 induced Kaiso localization at cell protrusion sites and in nucleus requires RhoH function in primary T cells of wildtype and Rhoh−/− mouse spleens. (A) T cells isolated from wildtype (WT) and Rhoh−/− mouse spleens were stimulated with SDF-1 (200ng/ml) for 30 seconds and stained with anti-Kaiso monoclonal antibody (red) and nuclear DAPI (blue). (B) Isolated T cells were stimulated with SDF-1 (200ng/ml) for 15 minutes and stained with anti-Kaiso monoclonal antibody (red) and nuclear DAPI (blue). Cell images were captured by Zeiss LSM 700 Laser Scanning Confocal-Microscope with 63X magnification and processed by ZEN software (Zeiss). Top row is WT and lower row is Rhoh−/− T cells.

Article Snippet: Finally, Kaiso or p120 catenin were detected with anti-Kaiso monoclonal antibody (Abcam, Cambridge, MA 1:500 dilution) or anti-p120 catenin antibody (BD Biosciences Transduction Laboratories, San Diego, CA) and HRP-coupled anti-mouse IgG antibody (1:2000, Cell Signaling Technology, Danvers, MA) using chemiluminescence (Cell Signaling Technology, Danvers, MA).

Techniques: Isolation, Staining, Microscopy, Software

Journal: Small GTPases

Article Title: RhoH participates in a multi-protein complex with the zinc finger protein kaiso that regulates both cytoskeletal structures and chemokine-induced T cells

doi: 10.1080/21541248.2016.1220780

Figure Lengend Snippet: RhoH is required for TCR activation-induced Kaiso nuclear localization. (A) Co-localization of RhoH and Kaiso in nucleus after TCR activation. Jurkat cells were plated on anti-CD3 monoclonal antibody coated wells for 15min, fixed and stained with polyclonal anti-RhoH antibody (green), monoclonal anti-Kaiso antibody (red) and nuclear staining with DAPI (blue). Cell Images were captured by Zeiss LSM 700 Laser Scanning Confocal-Microscope with 63X magnification and processed by ZEN software (Zeiss). RhoH and Kaiso co-localized in the nucleus indicated by arrowhead vs. arrow. Represenative images of 3 independent experiments are shown. (B) RhoH knockdown is associated with the loss of Kaiso nuclear localization after TCR activation. Jurkat cells transduced with control or RhoH shRNA were plated on anti-CD3 monoclobnal antibody coated wells for 15min and then stained with monoclonal anti-Kaiso antibody (red), RhoH (purple) and nuclear DAPI staining (blue). Cell images were captured by Zeiss LSM 700 Laser Scanning Confocal-Microscope with 63X magnification and processed by ZEN software (Zeiss). The loss of RhoH and Kaiso co-localization in the nucleus indicated by arrowhead vs. arrow. The images were analyzed in a Z axis single plane with 0.9um thickness to address Kaiso nuclear localization.

Article Snippet: Finally, Kaiso or p120 catenin were detected with anti-Kaiso monoclonal antibody (Abcam, Cambridge, MA 1:500 dilution) or anti-p120 catenin antibody (BD Biosciences Transduction Laboratories, San Diego, CA) and HRP-coupled anti-mouse IgG antibody (1:2000, Cell Signaling Technology, Danvers, MA) using chemiluminescence (Cell Signaling Technology, Danvers, MA).

Techniques: Activation Assay, Staining, Microscopy, Software, Transduction, shRNA