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Files in this Data Supplement:
Fig. S1. Co-immunolocalisation of SMC4 and CAP-D2. (A) Schematic representation of the protein region used to generate the CAP-D2 antibody (amino acids 951 to 1162 amino acid residues). Immunoblot analysis against cell extract with the CAP-D2 antibody revealed that the antibody recognises a band of the expected size (~148 kDa, asterisk) using three different bleeds (1,2 and 3) in a dilution of 1:10,000. The band was absent from the pre-immune lane (1=1:1000, 2=1:5000, and 3=1:10,000 dilutions). (B) Schematic representation of the protein region used to generate the CAP-D3 peptide antibody (15 terminal amino acid residues were used). Immunoblot analysis against cell extract with the CAP-D3 antibody revealed that the antibody recognises a prominent band of about 80 kDa (asterisk). The band was absent from the pre-immune lane. (C) Co-immunolocalisation of CAP-D2 and SMC4. Cells were cytospun onto poly-L-lysine slides, extracted during fixation and stained for SMC4 (red), CAP-D2 (green), and DNA (blue). The two proteins co-localise during prometaphase. SMC4 dissociates from chromosomes as they decondense during anaphase, while CAP-D2 persists throughout the chromosomes. In telophase SMC4 is diffuse in the cytoplasm and the nucleus, while CAP-D2 is still tightly associated with chromosomes. Scale bar: 10 mm.
Fig. S2. Quantification of effects on cells after CAP-D2 RNAi. (A) Growth curve showing the number of cells at different time points after CAP-D2 dsRNA treatment. Cells grew more slowly, plateaued at 72 hours, and did not change significantly after that time. (B) The percentage of mitotic cells in control and CAP-D2 RNAi cells. The percentage of mitotic cells increased two- to threefold in the CAP-D2 RNAi between 36 and 72 hours (6.7% versus 2.2% at 48 hours). (C) The percentage of abnormal mitotic cells in control and CAP-D2 RNAi cells. The majority of mitotic cells are abnormal 36 hours and later after dsRNAi treatment. (D) Histogram showing the percentage of cells in prometaphase after staining for Cyclin B/P-H3/a-tubulin in control and CAP-D2 RNAi cells. Cells delay in prometaphase in the CAP-D2-depleted cells. (E) Histogram showing the percentage of cells in anaphase after staining for Cyclin B/P-H3/a-tubulin, in control and CAP-D2 RNAi cells. The anaphase index in CAP-D2 RNAi cells is lower than in control cells. (F) Immunoblotting of control and CAP-D2-depleted extracts for topo II shows that depletion of CAP-D2 does not affect the level of topo II. a-tubulin was used as a loading control. C, control dsRNA; –, no RNA; D, CAP-D2 dsRNA.
Fig. S3. CAP-D3 localisation in mitotic control and CAP-D2-depleted cells. A. Immunolocalisation of CAP-D3 in S2 cells. Cells were cytospun onto poly-L-lysine slides, extracted during fixation and stained for CAP-D3 (green), CID (red) and DNA (blue). In control cells, CAP-D3 localises at the centromeres and partially overlaps with CID at prometaphase, metaphase and anaphase. The antibody gives a non-specific centrosomal staining as well. B. CAP-D2 RNAi cells 72 hours after treatment. Cells were cytospun onto poly-L-lysine slides, extracted during fixation and stained for CAP-D3 (green), CID (red) and DNA (blue). CAP-D3 protein still localises to CID-positive regions in the CAP-D2-depleted cells. Scale bar: 10 mm. C. Immunoblotting of control, CAP-D2-depleted, topo II-depleted and CAP-D2/topo II doubly depleted cell extracts with the CAP-D3 antibody. CAP-D3 levels are unaffected in either the single or the double depletions. a-tubulin was used as a loading control. C, control dsRNA; –, no RNA; D, CAP-D2 dsRNA; T, topo II dsRNA; D/T, CAP-D2/topo II double dsRNA.
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