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Fig. 3. CAP-D2 dsRNA-mediated interference in Drosophila S2 cells results in abnormal chromosome morphology and segregation. (A) Immunoblot showing the depletion of CAP-D2 after treatment with dsRNA. 5 x10⁵ cells were loaded per lane. Lanes: C, control dsRNA; –, no dsRNA; D, CAP-D2 dsRNA. CAP-D2 levels start to decrease as early as 48 hours and the protein is undetectable at 96 and 120 hours. -tubulin was used as a loading control. (B,C) Immunofluorescence analysis of control and CAP-D2 RNAi cells. Cells were cytospun onto poly-L-lysine slides 65 hours after treatment and stained for CAP-D2 (green), CID (red), and DNA (blue). (C) CAP-D2-depleted cells have no CAP-D2 on condensed chromosomes and exhibit abnormal chromosome morphology. Sister chromatids fail to resolve and segregate properly, and chromosomes are not properly aligned on the metaphase plate. Centromeres appear stretched. (D,E) Hypotonic treatment of control (D) and CAP-D2-depleted (E) cells 65 hours after dsRNA treatment shows that chromatid resolution is defective in the CAP-D2-depleted cells. Cells were hypotonically treated, centrifuged onto poly-L-lysine slides, fixed and stained for CAP-D2 (red), P-H3 (green) and DNA (blue). Note that individual chromatids are not readily apparent after depletion of CAP-D2. Scale bar: 10 µm.

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