Cell lines originating from other species were Ptk-2, L929, HL-1 and MDCK cells. was based on, respectively, CD-Search  and InterPro  analyses. Other conserved regions, CD1, CD2 and NLS, are indicated by yellow background. The novel Spindle-Associated domains 1 and 2 (SA1 and SA2) are highlighted in pink. See also Fig. 1 for domain organization. References : Maglott D, Ostell J, Pruitt KD, Tatusova T (2007) Entrez Gene: gene-centered information at NCBI. Nucleic Acids Res 35: Methacholine chloride D26-31. : Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947C2948. : Marchler-Bauer A, Bryant SH (2004) CD-Search: protein domain annotations on the fly. Nucleic Acids Res 32: W327-331. : Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, et al. (2009) InterPro: the integrative protein signature database. Nucleic Acids Res 37: D211-215.(0.18 MB PDF) pone.0009203.s001.pdf (175K) GUID:?4F30EC7D-A123-41D4-A07F-4FB8CF6EE412 Figure S2: The GFP-tagged Kaiso fragment K4x causes formation of protrusions during interphase. HEK293 cells are transfected with construct GFP-K4x. DNA was stained with DAPI, and cells were imaged with a Zeiss Axiophot microscope (100 objective lens).(0.18 MB PDF) pone.0009203.s002.pdf (175K) GUID:?E0432230-7DAC-400F-B719-60778DBA803A Figure S3: The GFP-tagged Kaiso fragment K4z localizes at spindle material during mitosis. During metaphase (upper row) and anaphase (lower row) of transfected HEK293 cells, the Kaiso fragment K4z concentrates at the centrosomes (arrowheads) and the minus ends of the spindle microtubules (arrows). DNA was stained with DAPI and cells were imaged with a Leica DM IRE2 microscope (63 objective lens).(1.59 MB PDF) pone.0009203.s003.pdf (1.5M) GUID:?813B94DD-D66F-4259-B11C-1B03F49AE415 Figure S4: The GFP-tagged Kaiso fragments K4a and K4b localize at the spindle material during mitosis. In transfected HEK293 cells the K4a (upper row) and K4b (lower row) Kaiso fragments both localize at the spindle microtubules (arrows) and at the centrosomes (arrowheads). DNA was stained with DAPI and cells were imaged with a Zeiss Axiophot microscope (100 objective lens).(0.70 MB PDF) pone.0009203.s004.pdf (684K) GUID:?DB070474-6742-444D-AF90-539A0D356794 Movie S1: (3.41 MB AVI) pone.0009203.s005.avi (3.2M) GUID:?3039B0CF-29F2-4440-9BB0-B126B4BBAE93 Abstract Kaiso is a BTB/POZ zinc finger protein known as a transcriptional repressor. It was originally identified through its association with the Armadillo protein p120ctn. Subcellular localization of Kaiso in cell lines and in normal and cancerous human tissues revealed that its expression is not restricted to the nucleus. In the present study we monitored Kaiso’s subcellular localization during the cell cycle and found the following: (1) during interphase, Kaiso is located not only in the nucleus, but also on microtubular structures, including the centrosome; (2) at metaphase, it is present at the centrosomes and on the spindle microtubules; (3) during telophase, it accumulates at the midbody. We found that Kaiso is a genuine PCM component that belongs to a pericentrin molecular Methacholine chloride complex. We analyzed the functions of different domains of Kaiso by visualizing the subcellular distribution of GFP-tagged Kaiso fragments throughout the cell cycle. Our results indicate CLEC4M that two domains are responsible for targeting Kaiso to the centrosomes and microtubules. The first domain, designated SA1 for spindle-associated domain 1, is located in the center of the Kaiso protein and localizes Methacholine chloride at the spindle microtubules and centrosomes; the second domain, SA2, is an evolutionarily conserved domain situated just before the zinc finger domain and might be responsible for localizing Kaiso towards the centrosomal region. Constructs containing both SA domains and Kaiso’s aminoterminal BTB/POZ domain triggered the formation of abnormal centrosomes. We also observed that overexpression of longer or full-length Kaiso constructs led to mitotic cell arrest and frequent cell death. Knockdown of Kaiso accelerated cell proliferation. Our data reveal a new target for Kaiso at the centrosomes and spindle microtubules during mitosis. They also strongly imply that Kaiso’s function as a transcriptional regulator might be linked to the control of the cell cycle and.