FIG 11.

Sld5-depleted centrosomes fragment due to CENP-E- and Kid-mediated forces. (A) HeLa cells transfected with control GL2 or CENP-E siRNA were immunoblotted with anti-CENP-E antibody to confirm RNAi depletion. The numbers indicate levels of CENP-E protein relative to control GL2 siRNA-transfected cells. (B and C) GL2 or CENP-E siRNA-transfected samples were costained for α-tubulin (green) and γ-tubulin (red), whereas TOTO-3 was used to stain the nucleus (pseudocolor blue). The arrowheads point to unaligned chromosomes observed in CENP-E-depleted samples. Quantification of the congression defects is shown in panel C. (D) HeLa cells were transfected with control GL2, SLD5, or CENP-E siRNA as indicated, with the combined concentration brought to 80 nM with GL2 siRNA. The cells were fixed and costained for α-tubulin (green) and γ-tubulin (red), whereas TOTO-3 was used to stain the nucleus (pseudocolor blue). (E) The transfected cells were immunoblotted with anti-Sld5 antibody to confirm RNAi depletion. The numbers indicate levels of Sld5 relative to control GL2 siRNA-transfected cells. (F) Decrease of CENP-E mRNA confirmed by reverse transcriptase PCR. The numbers indicate the CENP-E mRNA levels following siRNA-mediated depletion relative to control GL2 siRNA-transfected cells. BMG served as the internal RNA-loading control. (G and H) HeLa cells transfected with control GL2, SLD5, or CENP-E siRNA, as described for panel D, were stained with anti-CENP-E antibody to confirm RNAi depletion. Quantification of the spindle pole defects is shown in panel H. (I) HeLa cells transfected with different siRNAs, as indicated, were surveyed to identify mitotic cells with low levels of Cep170 or pericentrin staining, which was confirmed by NIS Elements software for multiple examples to be less than 50% of the mean intensity of the signal observed in control cells. (J and K) HeLa cells transfected with control GL2, SLD5, or KID siRNA, as indicated, with the combined concentration brought to 80 nM with GL2 siRNA. The cells were fixed and costained for α-tubulin (green) and γ-tubulin (red) and for DNA with DAPI (blue). Note that inhibition of KID activity resulted in multiple chromosome congression defects. Quantification of the spindle pole defects is shown in panel K. (L) The transfected samples were immunoblotted with anti-Sld5 antibody to confirm RNAi depletion. The numbers indicate levels of Sld5 protein in different samples relative to control GL2 siRNA-transfected cells. (M) Decrease of KID mRNA confirmed by reverse transcriptase PCR. The numbers indicate the KID mRNA levels following siRNA-mediated depletion relative to control GL2-transfected cells. BMG served as the internal RNA-loading control. Quantification of data is represented as the means and SD of the results of two independent experiments, with more than 20 cells analyzed in each sample. *, P < 0.05. LC, loading control showing equal protein loads in different lanes. Scale bars, 10 μm.