Characterization of actin, dynein, and LGN perturbations. (A) Representative images of MCF10A cells transfected with Lifeact-mEmerald for 48 h, labeled with SiR-tubulin, and treated with 500 nM LatA or an equivalent volume of DMSO for 30 min. Projection images at right represent maximum intensity projections of 5 planes spaced 0.3 µm apart, showing the regions adjacent to (low) and far from (high) the coverslip. Channels are not scaled equally between the DMSO and LatA examples, to account for differences in Lifeact-mEmerald expression level. Scale bars = 5 µm. (B) Western blot of dynein intermediate chain levels in MCF10A cells transfected with siRNA targeting luciferase (siCtrl) or dynein heavy chain for 48 h. Depletion of dynein intermediate chain is correlated with dynein heavy chain depletion (Levy and Holzbaur, 2008). GAPDH is shown as a loading control. (C) Western blot of LGN levels in MCF10A cells transfected with siRNA targeting luciferase (siCtrl) or LGN for 48 h. GAPDH is shown as a loading control. (D) Maximum intensity projections of live MCF10A cells transfected with siRNA targeting luciferase (siCtrl, left) or dynein heavy chain (siDHC, right) for 48 h and labeled with SiR-tubulin. Yellow arrows indicate metaphase spindles with abnormal morphology after dynein knockdown. Images are not scaled equally. Scale bars = 5 µm. Source data are available for this figure: SourceData FS3.