Figure 4.

Disrupted astrin function and premature loss of sister chromatid cohesion in kinastrin-depleted or overexpressing cells. (A) HeLa Flp-in TRex Myc-kinastrin and HeLa Flp-in TRex Myc-astrin cells were mock treated or treated with 1 µg/ml doxycycline for 24 h, then stained for Myc, astrin, kinastrin, and tubulin or harvested for Western blotting with the indicated antibodies. Transfected kinastrin conforms to the longest splice variant of C15ORF23. DNA was stained with DAPI. Cells with different expression levels of Myc-kinastrin or Myc-astrin, respectively, are shown. Bar, 10 µm. (B) HeLa Flp-in TRex Myc-kinastrin cells were mock treated and mitotically arrested with 100 ng/ml nocodazole for 14 h or induced with 1 µg/ml doxycycline for 24 h. Chromosome spreads were prepared and the percentage of cells with separated sister chromatids was assessed. (C) HeLa Flp-in TRex Myc-kinastrin cells were treated as in A and stained for centrin-3 and tubulin. The percentages of spindle poles with zero, one, two, more than two, or markedly separated centrioles are plotted in the bar graph. (D) HeLa S3 cells treated with control, astrin, or kinastrin siRNA oligos for 48 or 72 h (kinastrin) were stained as in C. Centriole numbers at the spindle poles are plotted in the bar graph. (E) HeLa S3 cells were transfected with control, astrin, kinastrin, Sgo1, Sgo2, or HURP siRNA duplexes for 48 or 72 h (kinastrin), harvested by mitotic shake-off (control cells were treated with 100 ng/ml nocodazole for 14 h before harvest), and processed for chromosome spreads. 100 cells were counted from each condition (n = 3; top graph, representative images are shown on the left). Depletion of separase in addition to astrin or kinastrin significantly lowered the amount of cells with separated sister chromatids (middle graph). The addition of nocodazole to astrin- or kinastrin-depleted cells released from a thymidine block significantly reduced the amount of premature loss of sister chromatid cohesion (bottom graph). (F) HeLa cells arrested in mitosis with nocodazole, released from the nocodazole block, or treated with siRNAs targeting CenpE (48 h) or kinastrin (72 h) were harvested by mitotic shake-off (except nocodazole release), then lysed and blotted with the indicated antibodies. Note that kinastrin-depleted cells have cyclin B1 and securin levels lower than nocodazole-arrested cells but comparable to CenpE-depleted cells. The separase cleavage fragment, however, only appears in kinastrin-depleted cells. (G) Separase immunoprecipitations from HeLa cells depleted of astrin or kinasterin for 48 or 72 h were blotted as indicated. All error bars indicate the standard error of the mean. Numbers next to gel blots indicate molecular mass in kilodaltons.