File S1 - CENP-W Plays a Role in Maintaining Bipolar Spindle Structure

File contains Figures S1–S6. Figure S1. Cohesion fatigue is unlikely to be the main mechanism driving multipolarity in CENP-W depleted cells. (A) Representative panel of HeLa H2B-GFP cells depleted of CENP-W for 48 h and stained with ACA (blue) and Zwint-1 (red) for centromere and kinetochore localization, respectively. The vast number of scattered chromosomes contain clearly visible coupled centromeres (box 2) while some display split sister kinetochores (box 1). (B) Representative panel of GFP-CENP-W HeLa cells depleted of CENP-W for 48 h and stained for pericentrin (green) and centrin-3 (red), red arrows indicate the appearance of non-centriolar pole, which can originate as a consequence of spindle pole defects associated with centriole disengagement (cell #1) or mechanical disruption of the pole with normal paired centrioles (cell #2). Figure S2. Depletion of CENP-W protein in GFP-CENP-W HeLa cells. (A) HeLa cells expressing GFP-CENP-W were transfected with pooled siRNA for CENP-W (CW), CENP-T (CT) and non-targeting (NT) control. Samples harvested at indicated times were Western blotted against GFP and CENP-W for detection of a tagged and endogenous protein, respectively. Numbers represent relative protein abundance, measured by quantification of chemiluminescence signal and normalized to tubulin. (B) Representative image of GFP-CENP-W (green) cells, depleted for CENP-W and stained with DAPI (blue), upper panel. Quantitative measurements of the mean (+SEM) GFP fluorescence signal intensities of the mitotic prometaphases and metaphases cells, following siRNA transfection, lower panel. Values plotted were background corrected. *** p (PDF)