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. 2012 Jun 29;287(33):27670–27681. doi: 10.1074/jbc.M112.378968

FIGURE 1.

FIGURE 1.

Depletion of Fry causes chromosome misalignment and multipolar spindle formation. A, immunoblot analysis of endogenous Fry. HeLa cells transfected with control or Fry siRNA were cultured for 60 h, and lysates were immunoblotted (IB) with anti-Fry antibody. B, Fry depletion causes chromosome misalignment and multipolar spindle formation. HeLa cells transfected with control or Fry siRNA were cultured for 12 h in growth medium and then for 24 h in thymidine-containing medium. They were then released from thymidine arrest for 12 h before being fixed and stained for α-tubulin (red). DNA was stained with DAPI (blue). Scale bar = 10 μm. The arrow indicates misaligned chromosomes. The arrowheads indicate multiple spindle poles. C, percentages of cells with misaligned chromosomes and multipolar spindles. Data are means ± S.E. of triplicate experiments (more than 100 cells were scored for each experiment). *, p < 0.05; **, p < 0.01. D, Fry depletion causes extra centrosomal foci in mitosis. HeLa cells transfected with control or Fry siRNA were synchronized as in B and stained for γ-tubulin (red) and pericentrin (green). DNA was stained with DAPI (blue). Scale bar = 10 μm. E, percentages of cells with > 2 γ-tubulin-positive foci in control or Fry siRNA-treated HeLa cells in interphase or mitosis. F, percentages of cells with three, four, and more than four γ-tubulin-positive foci in control or Fry siRNA-treated HeLa cells in mitosis. In E and F, data are means ± S.E. of triplicate experiments (more than 100 cells for each experiment). *, p < 0.05; **, p < 0.01. G, Fry depletion causes centriole splitting. HeLa cells transfected with control or Fry siRNA were synchronized as in B. Cells were permeabilized, and then fixed and stained for centrin (green) and γ-tubulin (red). DNA was stained with DAPI (blue). Magnified images of numbered boxes are shown. Scale bars = 5 μm. H, quantitative analysis of spindle poles with no, one, and two centrioles in control or Fry siRNA-treated HeLa cells. Data are the means ± S.E. of triplicate experiments (more than 50 cells were scored for each experiment). *, p < 0.05.