Extended Data Figure 8 : KNL1 (also known as CASC5) is not required for a stabilized MCC to inhibit anaphase.

From: The mitotic checkpoint complex binds a second CDC20 to inhibit active APC/C

Extended Data Figure 8

a, HeLa cells expressing MCCM2 in Fig. 4a were analysed by immunostaining. The cells were stained with anti-GFP, anti-MAD2, anti-ACA and Hoechst 33342, and representative images of prometaphase and metaphase cells from two independent biological replicates are shown. Scale bar, 5 µm. b, The time from NEBD to anaphase in Fig. 4a was plotted against the intensity of mCherry–GBP–CDC20 (left) or the ratio of Venus–MAD2 to mCherry–GBP–CDC20 (right). The ratio of Venus–MAD2 to mCherry–GBP–CDC20 was calibrated by measuring fluorescence intensity of a mCherry–GBP–Venus fusion protein in HeLa cells. ce, MCCM2 delays anaphase when KNL1 is depleted. c, HeLa cells were treated with siRNA against KNL1 for 72 h and total cell extracts were analysed by quantitative immunoblotting with the indicated antibodies. d, HeLa cells treated as in c were analysed by immunostaining. The cells were stained with anti-CDC20, anti-BUBR1, anti-ACA and Hoechst 33342, and representative images of early prometaphase from two independent biological replicates are shown. Scale bar, 10 µm. e, HeLa cell lines stably expressing mCherry–GBP–CDC20 and an inducible 3×Flag–Venus–MAD2 (expressed from a tetracyclin-inducible promoter) were treated with siRNA against KNL1 as in c. Progression through mitosis was analysed in the presence (+Tet) or absence (−Tet) of tetracyclin, and analysed as in Fig. 4b. n, number of cells from two independent biological replicates.