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. 2019 May 21;218(7):2150–2168. doi: 10.1083/jcb.201805044

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© 2019 David et al.

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Figure 2. — Most MT plus ends in metaphase spindles are generated in an Augmin-dependent manner. (A) HAUS6 protein levels analyzed by Western blotting in HeLa cells transfected with control, HAUS4-targeting, or HAUS6-targeting siRNAs. (B) HAUS6 and α-tubulin visualized by immunofluorescence in metaphase spindles of HeLa cells transfected with control or HAUS6-targeting siRNAs. (C and D) 3D lattice light-sheet microscopy of HeLa cells expressing EB3-EGFP (green) and mCherry-CENPA (magenta), transfected with either nontargeting control siRNAs (C) or siRNAs targeting HAUS6 (D). 2.5-min videos of metaphase cells were acquired at 1 s/frame. Deconvolved images are shown. (E) EB3-EGFP fluorescence intensities measured in interpolar regions of the spindle as in Fig. 1 I for control and HAUS6 RNAi cells (n = 7 and 11 cells, respectively, collected in two independent experiments). (F) First derivative of EB3-EGFP fluorescence profiles shown in E, indicating rate of increase for MT plus end numbers. Lines and shaded areas denote mean ± SD, respectively. Scale bars, 10 µm. AU, arbitrary units.