FIGURE 2:

Centrosome clustering is compromised in the absence of cortical dynein activity. Representative fixed-cell images of cell lines with extra centrosomes, through overexpression of PLK4 (A: RPE indPLK4), DCB to induce cytokinesis failure (C: RPE DCB), or preexisting centrosome amplification (E: MDA-MB-231). Cells were treated with the dynein inhibitor dynarrestin, a nontargeting siRNA (siCtl), or a LGN or Afadin-targeting siRNA (siLGN or siAfadin) to disrupt dynein localization at the cortex (see Supplemental Figure S1 for experimental setup and confirmation of Afadin and LGN depletion). Antibodies specific for α-tubulin (green), centrin-2 (red), and DAPI (blue) to detect DNA were utilized to assess mitotic stage and spindle structure. The white asterisks denote spindle poles, insets represent 4× enlargements of individual centrosomes, and scale bars are 5 µm. Quantification of the percentage of mitotic cells (preanaphase) exhibiting clustered bipolar spindles is given: (B: RPE indPLK4), (D: RPE DCB), and (F: MDA-MB-231). The analysis was performed on at least 50 cells per condition from three biological replicates, and error bars are SD. Significance was determined via a Student’s t test when comparing two conditions and a one-way ANOVA with Dunnett’s test for multiple comparisons when comparing multiple conditions to one control; *p < 0.05, **p < 0.01.