We then directly examined whether the H3pT3\dependent recruitment of CPC to centromeres is required for the protection of centromeric cohesion. Taken together, Squalamine lactate these results indicate a kinase\dependent role for Haspin in antagonizing Wapl and protecting centromeric cohesion in mitosis. = 3, unpaired = 2). D The indicated cell lines were exposed to MG132, then fixed at the indicated Squalamine lactate time points to stain DNA. The percentage of mitotic cells with near\standard metaphase plate (< 3 misaligned chromosomes) was determined in over 300 cells (= 3, two\way ANOVA). E Cells were exposed to MG132 for 8 h. Using mitotic chromosome spreads, the percentage of cells with PCS was determined in over 70 cells. F Cells were treated with nocodazole for 3 h. Mitotic chromosome spreads were stained with CENP\C antibodies and DAPI. The inter\KT distance was measured on over 300 chromosomes in over 10 cells (unpaired = 3, two\way RNF55 ANOVA). J Cells were treated as in (I). At 5 h after STLC washout, mitotic cells were collected to prepare chromosome spreads; then, the percentage of cells with PCS was determined in over 50 cells. Data information: Means and SDs are shown (B, D, F, G, and I). See also Fig EV1. Open in a separate window Figure EV1 Haspin\KD contributes to Wapl inhibition and centromeric cohesion protection in mitosis (related to Fig ?Fig11) A Asynchronous cells of HeLa, clone D2 with or without stable overexpression of SFB\Haspin, were lysed and immunoblotted with the indicated antibodies. Note that these blots, which were recently published 37, are displayed here to indicate the overexpression of SFB\Haspin. B The indicated stable cell lines were transfected with control or Sgo1 siRNA. After 36 h, cells were exposed to nocodazole for 3 h. Mitotic chromosome spreads were immunostained (related to Fig ?Fig11C). C, D Haspin\KO cells stably expressing Haspin\GFP were transfected with control or Pds5B siRNA. After 48 h, cells were exposed to nocodazole for 3 h to prepare mitotic chromosome spreads and were immunostained with antibodies for GFP or CENP\C (C). Asynchronous cell lysates were subjected to immunoblotting (D). E Lysates prepared from asynchronous Haspin\KO cells stably expressing SFB\Haspin or Haspin\N50\GFP were immunoblotted with the indicated antibodies. F The indicated stable cell lines were exposed to MG132, then fixed at the indicated time points to stain DNA. The percentage of mitotic cells with near\standard metaphase plate (< 3 misaligned chromosomes) was determined in over 100 cells in each condition. Means and ranges are shown in cells except for HeLa (= 2). G Haspin\KO cells transiently expressing CB\GFP or Squalamine lactate the indicated CB\Haspin\GFP (N50, KD or mutants) fusion proteins were exposed to nocodazole for 3 h. Mitotic cells were cytospun onto coverslips, fixed, and immunostained with antibodies for H3pT3 or CENP\C to measure the inter\KT distance (see Fig ?Fig1G).1G). Example images are shown. Data information: scale bars, 10 m. Cells with chromosomal instability and weak cohesion undergo PCS to various extents, particularly during prolonged metaphase with sustained bipolar kinetochore tension 48, 49. As we recently reported, HeLa\derived Haspin\knockout (KO) cells (clone D2 used in this study), which have weakened centromeric cohesion 37, were defective in maintaining chromosome bi\orientation and sister\chromatid cohesion during metaphase arrest induced by treatment with the proteasome Squalamine lactate inhibitor MG132 (Fig ?(Fig1D1D and E). Squalamine lactate These defects were partly rescued by stable overexpression of SFB\Haspin\N469, which is in line with the role of Haspin N\terminus in binding Pds5B and protecting centromeric cohesion 37. In addition, the inter\kinetochore (inter\KT) distance on chromosome spreads prepared from Haspin\KO cells arrested in mitosis with the spindle microtubule poison nocodazole was 20% further apart than the control, indicative of weakened centromeric cohesion. Compared to full\length SFB\Haspin, SFB\Haspin\N469 was impaired in restoring proper inter\KT.