Fig. 4. Chromosome arm and centromere resolution are compromised after CAP-D2 depletion. (A) Immunoblotting showing that the levels of DSA1 were not affected 72 hours after CAP-D2 RNAi treatment. Lanes: C, control dsRNA; –, no dsRNA; D, CAP-D2 dsRNA. -tubulin was used as a loading control. (B,C) Centromere resolution is disrupted in the CAP-D2-depleted cells. Control (B) and CAP-D2-depleted (C) cells were cytospun onto poly-L-lysine slides 72 hours after dsRNAi treatment, fixed and stained for DSA1 (green), CID (red) and DNA (blue). White boxes indicate regions shown at higher magnification in the panels on the right. The majority of prometaphase/metaphase cells had normal CID staining between sister chromatids (C1 and higher magnification), while a small percentage showed a slight stretching (C2 and higher magnification). Cells in anaphase showed severe centromere stretching on chromosomes (C3-5 and higher magnification). Scale bar: 10 µm. (D) The distance (in µm) between paired CID spots of cells in prometaphase/metaphase after staining for DSA1/CID/DAPI. The distance in CAP-D2-depleted cells is more than twofold higher than in control cells.