Supplementary Figure 4: CENP-E is targeted to the kinetochores by hMad1.

(a) HeLa cells treated with the indicated siRNAs were examined by immunoblot using the indicated antibodies. Note that hMad1-1 siRNA is less effective than hMad1 siRNA, which is used in Figs 6 and 7. (b) HeLa cells treated with control siRNA or hMad1-1 siRNA were arrested in metaphase by MG132 and examined for chromosome alignment as in Fig. 6a. Error bars, s.e.m. for n = 3 independent experiments. Statistical significances (t-test, two-tailed) were assessed (*P < 0.05). (c) HeLa cells treated with control siRNA or hMad1-1 siRNA arrested in mitosis by nocodazole and MG132 were examined by immunostaining (left). Localization was quantified as the ratio of fluorescence intensity of CENP-E to the value of CENP-C in 10 kinetochores from each cell (right). Error bars, s.e.m. for n = 9 cells for each. Statistical significances (t-test, two-tailed) were assessed (*P < 0.05). (d) HeLa cells treated with indicated siRNAs were arrested by Monastrol and released into MG132 after the washout of Monastrol. To prevent premature mitotic exits in hMad1-depleted cells treated with Monastrol, MG132 was added to the culture 1 h after the Monastrol addition. In this synchronous mitotic culture, alignment defects in hMad1-depleted cells appeared transiently only after 1 h after release but not anymore at 2 h. This contrasts to the results in CENP-E-depleted cells, in which alignment defects were persistent. Because CENP-E is only partly displaced from centromeres in hMad1-depleted cells (Fig. 7a), this residual CENP-E might finally complete the alignment. Over 30 cells were analysed for each. (e) HeLa cells expressing hMad1-3Flag-HA or hMad1-5A-3Flag-HA arrested in mitosis by nocodazole were examined by immunostaining (left). Localization was quantified as the ratio of fluorescence intensity of CENP-E to the value of CENP-C in 10 kinetochores from each cell (right). Error bars, s.e.m. for n = 7 cells for each. Statistical significances (t-test, two-tailed) were assessed (**P < 0.005). hMad1 overexpression enhanced the CENP-E accumulation at kinetochores in N-terminal motif-dependent manner. (f) hMad1-depleted HeLa cells expressing RNAi-resistant hMad1-3Flag-HA or hMad1-5A-3Flag-HA were examined for its nuclear envelope localization by immunostaining. Single sections of the images are shown. Uncropped images of blots are shown in Supplementary Fig. 8. Scale bars, 4 μm.