JCS02614 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Cenp-F is not required for spindle checkpoint function. HeLa cells were transfetced with either control siRNA duplexes (green), or ones designed to repress Cenp-F (yellow) and BubR1 (red). 48 hours post-transfection, the cells were exposed to nocodazole and the mitotic index determined by immunofluorescence imaging. BubR1 RNAi cultures show a reduced accumulation of mitotic cells, consistent with a spindle checkpoint defect. By contrast, Cenp-F RNAi cells accumulate in mitosis similar to controls, suggesting that the checkpoint is intact. Values represent the meanąs.e.m. of three independent experiments in which at least 1000 cells were counted.

• Supplemental Figure 2 -

  Fig. S2. Malorientations observed in Cenp-F-deficient cells. Immunofluorescence images taken from Fig. 6 showing a bioriented kinetochore pair from a control cell, and the four aberrant orientations (enlargements a-d) observed in two Cenp-F-deficient cells. (a) shows a merotelic orientation, (b and d) show single kinetochores and (c) shows a single kinetochore that attached to both poles. The interpretations are based on the 2D projections shown in Fig. 6 and scanning through individual Z-stacks.

• Supplemental Figure 3 -

  Fig. S3. Multinucleation is not prevalent following Cenp-F RNAi. Cells were transfected with siRNA duplexes to repress Lamin B1, Cenp-F and Bub1 were then fixed and analysed 48 hours later to determine the extent of multinucleation. Whereas multinucleated cells are prevalent following repression of Bub1, they are relatively rare in the Cenp-F RNAi culture.

• Supplemental Figure 4 -

  Fig. S4. Quantitation of microtubule intensity along spindle length. (a) Inverted fluorescence image of the control spindle shown in Fig. 8A, (b) a schematic representation thereof, and (c) diagram showing how tubulin fluorescence was measured along the spindle length.

• Movie 1 -

  Movie 1. Time-lapse movie of a control HeLa cell expressing GFP-histone H2B fusion protein showing progression through a normal mitosis.

• Movie 2 -

  Movie 2. Time-lapse movie of a Cenp-F RNAi HeLa cell expressing GFP-histone H2B fusion protein showing chromosome alignment failure and mitotic exit without chromosome segregation.

• Movie 3 -

  Movie 3. Time-lapse movie of a Cenp-F RNAi HeLa cell expressing GFP-histone H2B fusion protein showing chromosome alignment failure. Note that the chromosome movements appear to be small Brownian-type movements rather than coordinated P and AP movements.

• Movie 4 -

  Movie 4. Time-lapse movie of a Cenp-F RNAi HeLa cell expressing GFP-histone H2B fusion protein showing that, whereas some chromosomes appear aligned on a tripolar spindle, many remain clustered near the spindle poles. Note that the chromosome movements appear to be a result of movement of the entire chromosome mass or the whole cell rather than coordinated movements of individual chromosomes.