FIGURE 5:

Stabilization of K-MTs improves bipolar spindle maintenance in RPE-1 cells after Eg5 inhibition. (A) Low concentrations of Taxol prevent spindle collapse in RPE-1 cells exposed to STLC. Representative images of RPE-1 cells treated with 2.5 nM Taxol plus MG-DMSO or MG-STLC as in Figure 1A. Tubulin (green) and centrin (magenta) were detected by immunostaining. DNA (blue) was counterstained with Hoechst 33342. LUTs were scaled identically. Scale bar, 5 μm. (B) Quantification of spindle geometries after MG-STLC treatment in RPE-1 cells treated with or without 2.5 nM Taxol. Data represent the mean ± SEM; n ≥ 300 cells from three experiments. *p < 0.001. (C) At 2.5 nM, Taxol does not prevent mitotic progression in RPE-1 cells. RPE-1 cells treated with or without 2.5 nM Taxol were filmed by differential interference contrast, and the time from nuclear envelope breakdown (NEBD) to anaphase onset (AO) was quantified. n ≥ 68 cells from two experiments. (D) Live-cell imaging of an RPE-1 spindle challenged with STLC in the presence of 2.5 nM Taxol. Still images of an RPE-1 cell expressing mCherry-tubulin treated first with 2.5 nM Taxol and 5 μM MG-132 for 100 min and then treated with 2.5 nM Taxol, 5 μM MG-132, and 10 μM STLC. Time is indicated in minutes and is relative to STLC addition. Scale bar, 5 μm. (E) At 2.5 nM, Taxol promotes K-MT survival of nocodazole shock in RPE-1 cells. Representative images of RPE-1 cells treated with nocodazole as in Figure 2 in the presence of 2.5 nM Taxol. Tubulin (green) and centrin (magenta) were detected by immunostaining. DNA (blue) was counterstained with Hoechst 33342. Scale bar, 5 μm. (F) Quantification of residual MT polymer levels of RPE-1 cells with or without 2.5 nM Taxol after nocodazole treatment as in E. Data represent the mean ± SEM; n ≥ 150 cells from four experiments. *p < 0.001. (G) The protective effect of low-dose Taxol on STLC depends on K-MTs. RPE-1 cells transfected with control or Nuf2 siRNA were treated with MG-DMSO or MG-STLC in the presence of 2.5 nM Taxol. Representative spindle geometries are shown. LUTs are scaled identically. Scale bar, 5 μm. (H) Quantification of spindle geometries after treatment as in G. Data represent the mean ± SEM; n ≥ 95 cells from three experiments. *p < 0.005.