SEARCH

SEARCH BY CITATION

This article includes Supplementary Material available from the authors upon request or via the Internet at http://www.interscience.wiley.com/jpages/0021-9541/suppmat .

FilenameFormatSizeDescription
jws-jcp.20725.fig1.tif2651KSupplementary figure 1. CSPP-egfp expression dependent localization in mitosis and induced phenotypical changes. CSPP-egfp expressing Hek293T cells were fixed 24h post-transfection and stained for centrosomes (anti--tubulin, red) and DNA (DRAQ5, blue) to analyze the localization of CSPP-egfp (green) at different expression levels by means of laser scanning confocal microscopy (A and B). Image acquisition settings (magnification, pinhole, excitation laser power and detector PMT voltage) were adjusted to right below egfp-signal saturation in CSPP-egfp high expressing cells and kept constant thereafter to allow quantitative comparison. High level CSPP-egfp expression induces formation of multiple asters in the absence of centrosome amplification, as judged by the presence of only two distinct g-tubulin staining foci. At the lowest detectable CSPP-egfp expression levels no phenotypical spindle abberations are observed (B). For improved visualization of the localization of CSPP-egfp the acquired CSPP-egfp image was contrast enhanced (stretched) (B, lower panel). CSPP-egfp localized to both centrosomes and spindle microtubules. Untransfected Hek293T cells served as control to ensure the specificity of the enhanced egfp signal (C).
jws-jcp.20725.fig2.tif2828KSupplementary figure 2. CSPP-L-egfp expression dependent loacalization in mitosis and induced phenotypical changes. CSPP-L-egfp expressing Hek293T cells were fixed 24h post-transfection and stained for DNA (DRAQ5, blue) and either or microtubules (anti--tubulin, red) or centrosomes (anti-α-tubulin, red) to analyze the localization of CSPP-egfp (green) at different expression levels by means of laser scanning confocal microscopy (A and B). Image acquisition is performed as described in supplementary figure 1. High level CSPP-L-egfp expression induces formation of monoplolar asters (A, cell to the lower left). At lower expression levels prometaphase intermediates are observed with separated spindle poles (A). At the lowest detectable CSPP-L-egfp expression levels no phenotypical spindle abberations are observed (B). For improved visualization of the localization of CSPP-egfp the acquired CSPP-egfp image was contrast enhanced (stretched) (B, lower panel). CSPP-L-egfp localized to both centrosomes and spindle microtubules.
jws-jcp.20725.fig3.tif1363KSupplementary figure 3. Control staining of Nocodazole treated transfectants (see Figure 4) for microtubule depolymerisation (alpha-tubulin, red; egfp-fusion proteins, green).
jws-jcp.20725.fig4.tif3461KSupplementary figure 4. Effect of CSPP(1-503)-egfp and CSPP-L(295-848)-egfp expression on microtubule organization in interphase. CSPP(1-503)-egfp (A) but not CSPP-L(295-848)-egfp (B) promotes over-stabilization of centrosome outgrowing microtubules in interphase cells (anti-α-tubulin, red; egfp-fusion proteins, green). Expression of neither of the constructs confers resistance to microtubule polymerization by Nocodoazole treatment (5μg/ml, 4h).

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.