FIGURE 1:

The ability of human cell lines to maintain spindle bipolarity in the absence of Eg5 activity varies. (A) Bipolar spindle collapse assay. Cells were arrested in mitosis for 90 min with 5 μM MG-132 and then treated for an additional 90 min with 5 μM MG-132 plus 10 μM STLC to inhibit Eg5 (“MG-STLC”) or with 5 μM MG-132 plus DMSO equal in volume to STLC (“MG-DMSO”), and fixed. (B, C) Representative images of HeLa (B) or RPE-1 (C) cells treated with MG-DMSO or MG-STLC. Tubulin (green) and centrin (magenta) were detected by immunostaining. DNA (blue) was counterstained with Hoechst 33342. Scale bar, 5 μm. (D) Quantification of spindle geometries after MG-STLC treatment as described in A. Data represent the mean ± SEM; n ≥ 300 cells from three experiments. (E) Quantification of spindle geometries after treatment with 10 μM STLC for 90 min without MG-132 treatment. Data represent the mean ± SEM; n ≥ 280 cells from three experiments. (F, G) Live imaging of HeLa and RPE-1 cell responses to STLC. Still images of HeLa (F) or RPE-1 (G) cells expressing mCherry-tubulin, arrested with 5 μM MG-132 for 100 min, and then treated with 5 μM MG-132 and 10 μM STLC. Time is indicated in minutes and is relative to STLC addition. Scale bar, 5 μm.