Sensors at Centrosomes Reveal Determinants of Local Separase Activity
Figure 3
Astrin (Spag5), Aki1 and Sgo1 depletion does not promote centriole disengagement.
(A) Immunoblot analysis of HeLa cells following astrin and sSgo1/Sgo1 depletion. α-Tubulin was used as loading control. (B) Depletions of Sgo1, astrin or Aki1 caused premature loss of sister chromatid cohesin. Chromosome spreads of HeLa cells are shown. Four fold enlargements on the right show the separated sister chromatids. Scale bar: 10 µm. (C) Representative images of HeLa cells stably expressing the Scc1 and kendrin sensors upon depletion of Sgo1, astrin or Aki1. The two-fold enlargements show the centrosomes. The relative mCherry/eGFP ratios upon siRNA treatment of cells are depicted on the right for the Scc1 (n>45) and kendrin (n>15) sensors. One-way ANOVA was used as statistical test (* represents p<0.05). Error bars are SD. Scale bar: 10 µm. (D) The percentages of multipolar spindles did not change in response to Sgo1 depletion in HeLa WT and HeLa H2B-RFP cells. The images on the right indicate examples of cells with multipolar and bipolar metaphase spindles. Scale bar: 10 µm. n = 30. Error bars indicate SD.