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. Author manuscript; available in PMC: 2009 Nov 25.
Published in final edited form as: Curr Biol. 2008 Nov 25;18(22):1778–1784. doi: 10.1016/j.cub.2008.08.012

Figure 1. Hec1 depletion from PtK1 cells results in mitotic defects.

Figure 1

(A) Average Hec1 kinetochore fluorescence intensity in Hec1 siRNA-transfected and mock-transfected cells (left). Cells transfected with Cy5-labeled Hec1 siRNA were identified and those with reduced levels of kinetochore-associated Hec1 were subjected to kinetochore fluorescence intensity analysis (n= 11 cells; 170 kinetochores). Kinetochores from mock-transfected cells were also analyzed (n= 13 cells; 198 kinetochores) and normalized to one. For one representative mock-transfected and one representative Cy5-labeled Hec1 siRNA-transfected cell, individual kinetochore intensities for all kinetochores within that cell are shown (right). The P value (asterisk) is <0.0001, as measured by Student's t-test.

(B) Images of mock-transfected and Hec1 siRNA-transfected cells. Cells were fixed 48 h post-transfection, immunostained with the indicated antibodies, imaged, and deconvolved. Projections of image stacks are shown. Overlay: tubulin (white), ACA (red), Hec1 (green).

(C) Quantification of end-on microtubule association with kinetochores (mock-transfected late prometaphase: n= 10 cells / 212 kinetochores; mock-transfected early-mid prometaphase: n= 10 cells / 208 kinetochores; Hec1 siRNA-transfected: n= 10 cells / 229 kinetochores).

(D) Quantification of inter-kinetochore distances, which were measured from ACA-centroid to ACA-centroid (mock-transfected prophase: n= 5 cells / 25 kinetochore pairs; mock-transfected metaphase: n= 14 cells / 61 kinetochore pairs; Hec1 siRNA-transfected prophase: n= 10 cells / 77 kinetochore pairs; Hec1 siRNA-transfected: n= 32 cells / 307 kinetochore pairs). The asterisk indicates a P value of <0.0005, as measured by Student's t-test (vs. mock-transfected metaphase).

(E) Mock-transfected and Hec1 siRNA-transfected cells were subjected to a cold-induced microtubule depolymerization assay, processed for immunofluorescence, immunostained with the indicated antibodies, imaged, and deconvolved. Projections of image stacks are shown.

(F) Time-lapse image stills of mock-transfected and Hec1 siRNA-transfected cells. Elaspsed time is indicated in min.

(G) Average time for progression through mitosis for mock-transfected (n=28 cells) and Hec1 siRNA-transfected (n= 86 cells) PtK1 cells. Time was scored from nuclear envelope breakdown (NEBD) to anaphase onset. Vertical line in all bar graphs indicates standard deviation.