Figure 7. Kinetochore–microtubule attachments are required for centrosome separation in prometaphase. (A) Immunoblot of an extract of HeLa cells 48 h after transfection with siRNA oligos against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or Nuf2. (B) Still images from videos of HeLa cells coexpressing H2B–monomeric RFP and tubulin-YFP. Cells were transfected with siRNA oligos against GAPDH or Nuf2 and 48 h later were arrested with STLC. Time is given in minutes relative to STLC washout. Images are maximum intensity projections of five confocal z sections spaced 2 µm apart. Green, tubulin; purple, histone H2B. (C) Frequency of spindles that remained monopolar 90 min after release from STLC in cells treated with siRNA to GAPDH or Nuf2. Error bars represent the mean and SEM of three separate experiments; n = 192 cells GAPDH siRNA; n = 228 cells Nuf2 siRNA; ***, P < 0.0001 by Fisher's exact test.
Figure 7.

Kinetochore–microtubule attachments are required for centrosome separation in prometaphase. (A) Immunoblot of an extract of HeLa cells 48 h after transfection with siRNA oligos against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or Nuf2. (B) Still images from videos of HeLa cells coexpressing H2B–monomeric RFP and tubulin-YFP. Cells were transfected with siRNA oligos against GAPDH or Nuf2 and 48 h later were arrested with STLC. Time is given in minutes relative to STLC washout. Images are maximum intensity projections of five confocal z sections spaced 2 µm apart. Green, tubulin; purple, histone H2B. (C) Frequency of spindles that remained monopolar 90 min after release from STLC in cells treated with siRNA to GAPDH or Nuf2. Error bars represent the mean and SEM of three separate experiments; n = 192 cells GAPDH siRNA; n = 228 cells Nuf2 siRNA; ***, P < 0.0001 by Fisher's exact test.

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