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Fig. 3. Double RNAi experiments suggest that the Drosophila Mob4 (Dmob4) phenotype is more similar to Asp than dynein or Ncd. (A) Representative mitotic spindle morphology after RNAi of the genes indicated. (-tubulin, red; Dgrip84, green; DNA, blue; bar, 5 µm.) (B) Quantitation of K fiber unfocusing in metaphase spindles. The relative mean width of K fiber minus ends – the distance between the minus ends of the outermost K fibers at each pole (blue line in inset) relative to control cells [average 2.08±0.03 µm (mean±s.d.)] – is shown after RNAi of the gene(s) indicated. RNAi of Mob4, Asp or Ncd induced significant increases in the focal width of K fibers relative to that of control cells, whereas Dhc64C had a minimal effect. Co-depletion of Dhc64C and Ncd had a strong synergistic effect, whereas the synergism in cells co-depleted of Dhc64C and either Asp or Mob4 was minimal. [Error bars indicate the s.d. of the average K fiber unfocusing distance measured in three independent experiments (n>45 spindles for each trial).] (C) Quantitation of centrosome detachment in the same spindles measured in (B). The distance of the gap between the centrosome (Dgrip84-staining foci that nucleate astral microtubules) and the minus end of the K fiber lying closest to the centrosome (green line in inset), relative to control cells [average 1.92±0.07 µm (mean± s.d)] is shown. RNAi of Mob4, Asp or Dhc64C, but not Ncd, caused an increase in centrosome detachment (statistically significant difference for Mob4 RNAi and control cells; P<0.0001). Co-depletion of Dhc64C and either Asp or Mob4 produced a synergistic increase in centrosome detachment, whereas no synergism was observed for co-depletion of Dhc64C and Ncd.

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