Supplemental Materials

This article contains the following supporting material:

• Fig7 Video 01 - A typical mitotic HEK293T cell co-expressing RFP-Nudel and H2B-GFP. An RFP image is presented in the first frame followed by GFP images recorded in 1.5-min intervals for 106.5 min. Display rate is 6 frames/s. Representative frames are shown in Figure 7C.
• Fig7 Video 02 - A typical mitotic HEK293T cell co-expressing RFP-Nudel^C36 and H2B-GFP. An RFP image is presented in the first frame followed by GFP images recorded in 1.5-min intervals for 180 min. Display rate is 6 frames/s. Representative frames are shown in Figure 7C.
• Fig7 Video 03 - A typical cell cotransfected with pTER and pEGFP-H2B. Two rounds of transfection were performed for pTER as described in Methods. GFP images were recorded at 1.5-min intervals for 106.5 min. Representative frames are shown in Figure 7D.
• Fig7 Video 04 - A typical cell cotransfected with pTER-Nudi and pEGFP-H2B. Two rounds of transfection were performed for pTER-Nudi as described in Methods. GFP images were recorded at 1.5-min intervals for 180 min. Representative frames are shown in Figure 7D.
• Figure S1 - Lis1 is not required for kinetochore localization of Nudel. (A) Kinetochore localization of Lis1-binding-defective GFP-Nudel^N20 mutant in HEK293T cells. (B) Typical nocodazole-treated cells expressing GFP-tagged Lis1 (panels 1-4) or Lis1N (panels 5-8). Arrows point to spindle poles and arrowheads indicate typical kinetochores. Scale bar, 10 μm.
• Figure S2 - Silencing ZW10 by RNAi. HEK293T cells were cotransfected with pEGFP-ZW10 and pTER-Luci (lane 1) or pTER-ZWi (lane 2) for 48 h. Immunoblotting was then performed to visualize the indicated proteins.
• Figure S3 - Specificity of antibody and RNAi construct against Nudel. (A) Antibody specificity. FLAG-Nudel (lane 1) and FLAG-NudE (lane 2) expressed in HEK293T cells were subjected to immunoblotting with the indicated antibodies. (B) Depletion of Nudel by RNAi. Vec, Con, or RNAi indicates transfection with pTER the vector, pTER-Luci as an additional control, or pTER-Nudi for repressing Nudel, respectively. HEK293T cells were transfected as described in Methods and collected at the indicated time for immunoblotting. Arrows point to Nudel and its phosphorylated forms (Yan et al., 2003). Arrowhead indicates a non-specific band.
• Figure S4 - Nudel^C36 overexpression attenuates kinetochore dynein, dynactin, Lis1, and CLIP-170. (A-B) HEK293T cells transiently expressing either GFP-Nudel or GFP-Nudel^C36 were treated with nocodazole (10 μg/ml) for 1 h prior to fixation. Transfectants in (B) are outlined. Arrowheads indicate representative kinetochores. Bar, 10 μm. (C) Relative kinetochore intensities of the indicated proteins. Total n is shown above each pair of histograms.
• Figure S5 - Effects of Nudel on spindle organization. (A) Typical MT organizations and NuMA distributions after transient expression of GFP-Nudel or GFP-Nudel^C36. (B) Typical MT staining in control (panels 1-4) or Nudel RNAi (panels 5-8) cells. Arrows indicate spindle poles. Scale bar, 10 μm.