A kinase‐dependent role for Haspin in antagonizing Wapl and protecting mitotic centromere cohesion AThe lysates from asynchronous Haspin‐KO cells stably.

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A kinase‐dependent role for Haspin in antagonizing Wapl and protecting mitotic centromere cohesion AThe lysates from asynchronous Haspin‐KO cells stably expressing the indicated Haspin‐GFP (WT or D707L/D709N) proteins were analyzed by immunoblotting. B–DHaspin‐KO clone D2 cells stably overexpressing Haspin‐GFP or Haspin‐D707L/D709N‐GFP (clone #2) were exposed to nocodazole for 3 h. Mitotic chromosome spreads were immunostained with antibodies for GFP, H3pT3, or CENP‐C and DAPI. Example images are shown in Fig A. The centromeric Haspin‐GFP/CENP‐C immunofluorescence intensity ratio on over 330 chromosomes in 10 cells was determined (B, unpaired t‐test). The inter‐KT distance was measured on over 500 chromosomes in 10 cells (C, unpaired t‐test). Means and ranges are shown for cells expressing Haspin‐GFP (WT or D707L/D709N) (D; n = 2). EThe indicated stable cell lines were exposed to MG132 for 8 h. Mitotic cells collected by shake‐off were used to prepare chromosome spreads and then were stained with CENP‐C antibodies and DAPI. Example images are shown (related to Fig D). FAsynchronous cells of HeLa, clone D2 with or without stable expression of SFB‐Haspin‐K511A were lysed and analyzed by immunoblotting. Note that the blots of Haspin and tubulin were previously shown . GThe indicated stable cell lines 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 250 chromosomes in 10 cells (unpaired t‐test). H6xHis‐Haspin‐KD (WT or K511A) was subjected to pulldown by GST or Wapl‐N‐GST followed by CBB staining. I, JHeLa cells were treated with nocodazole for 3 h; then, mitotic cells were collected and further treated for 1 h with MG132 and the indicated Haspin inhibitors (or DMSO as the vehicle control) in the continued presence of nocodazole. Mitotic cells were then cytospun onto coverslips and fixed for immunostaining. The inter‐KT distance was measured on over 840 chromosomes in 20 cells (I, unpaired t‐test). Example images are shown (J). K6xHis‐Haspin‐KD was subjected to pulldown by GST or Wapl‐N‐GST, in the presence of increasing concentrations of 5‐ITu, followed by CBB staining. L, MHeLa and the indicated stable cell lines were treated with nocodazole for 14 h; mitotic cells were collected. The samples were analyzed by immunoblotting twice with relatively long (L) and short (M) exposure times. Ex., exogenous survivin; En., endogenous survivin. N, OThe indicated cell lines were fixed and immunostained. The representative mitotic cells are shown (N). The Aurora B/centromere autoantigen immunofluorescence intensity ratio was determined on around 100 or 50 chromosomes in 10 cells (O, unpaired t‐test). PThe indicated cell lines were released from 3‐h treatment with STLC into MG132‐containing medium and then fixed at the indicated time points for DNA staining. The percentage of mitotic cells with near‐standard metaphase plate was determined in around 100 cells. QThe indicated cell lines 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 500 chromosomes in 15 cells (unpaired t‐test). Data information: Means and SDs are shown (B, C, G, I, O, and Q; n = 3). Scale bars, 10 μm. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO Rep, Volume: 19, Issue: 1, Pages: 43-56, First published: 14 November 2017, DOI: (10.15252/embr.201744737)