(A) Representative images show YFP-Astrin (WT or 4A) kinetochore intensities in HeLa cells depleted of endogenous Astrin and arrested using MG132. Cells were immunostained with antibodies against GFP and SKAP and CREST antisera and stained with DAPI for DNA. (B) Graph of percentage of Astrin sleeves or crescents at the outer-kinetochores of YFP-Astrin (WT or 4A), as indicated, in cells treated as in (A). Black bars and whiskers mark average value and standard deviation, respectively, from values across two experimental repeats. ‘*' indicates statistically significant differences. (C) Time-lapse images of HeLa FRT/TO cells treated with Astrin siRNA and expressing YFP-Astrin (WT or 4A), as indicated. Yellow triangle indicates the cell tracked; Yellow asterisks highlight prolonged delay between metaphase and anaphase. Cytoplasmic YFP signal was used to assess Nuclear Envelope BreakDown (NEBD), wide-field and YFP images were used to assess bipolar metaphase spindles undergoing anaphase (AO). (D) Cumulative graph of percentage of HeLa FRT/TO cells (as in C) that initiated NEBD (Nuclear Envelope Break-Down) and completed AO (Anaphase Onset) within time intervals indicated. n and T50 refer to cell numbers and AO time in 50% of cells, respectively. Astrin ∆70 and WT data are from Figure 2G. (E) Time-lapse images of HeLa FRT-TO YFP-Astrin (WT or 4A) siRNA-resistant cells, depleted of endogenous Astrin and arrested with MG132 (1 hr) before imaging. Green and magenta circles highlight Astrin-crescents. KT2 (magenta circle) in YFP-Astrin-4A expressing cell marks an Astrin-crescent gradually dimming into an Astrin-sleeve. Yellow asterisk refers to this blinking event. (F) Frequency graph of distribution of the number of blinking events (as in E) in kinetochores tracked for 60 s. KTs and n refers to the kinetochore numbers and blinking events, respectively. (G) Graph of average distance between Astrin-crescents of kinetochore pairs measured using time-lapse movies as in (E). Black bars and whiskers mark average value and standard deviation, respectively, across at least two experimental repeats. ‘*' indicates statistically significant differences. Scale as indicated.