Figure 2.

SAT III knockdown causes severe mitotic defects. (A) Cells transfected with SAT III–LNA probes targeting sense and antisense transcripts show severe mitotic defects with lagging chromosomes, whereas untreated cells or control cells transfected with scrambled LNA segregate properly. Cells were cotransfected with fluorescein-labeled dextran for selection of transfected cells. (B) Quantification of the properly dividing cells after SAT III depletion. Properly dividing cells in mock-transfected control (n = 104) were normalized to 1, and compared with scrambled LNA transfected (n = 49) and SAT III–LNA1 and LNA2 transfected cells (n = 115). (C) Live cell analysis of SAT III–depleted cells. S2 cells expressing GFP-labeled histone H2B and tubulin labeled with mCherry were transfected with SAT III–LNA probes, or with scrambled RNA (control). SAT III knockdown cells showed prolonged anaphase with lagging chromosomes that eventually formed micronuclei at the end of mitosis. (D) Quantification of properly dividing cells in C after SAT III depletion. Normal cell division in scrambled LNA transfected control (n = 18) was normalized to 1, and compared with SAT III–LNA1 and –LNA2 transfected cells (n = 24). (E) SAT III depletion causes severe mitotic defects in developing D. melanogaster embryos. Embryos from w¹¹¹⁸ flies were injected with scrambled control LNA probes or SAT III–specific probes. SAT III–depleted embryos formed anaphase bridges, and were unable to segregate chromosomes properly. (F) Zhr1 embryos display mitotic defects. All Zhr1 pre-gastrulation embryos in mitosis (n = 20) displayed major chromosome segregation defects, whereas control embryos (Oregon-R) divided normally (n = 16). The arrows indicate anaphase figures with chromosome bridges in preblastoderm embryos and syncytial embryos as well as missegregated DNA mass at the syncytial stage. Panels on the right are enlarged from the boxed regions. Bars: (A, C, and F) 5 µm; (E, left) 10 µm; (E, right) 5 µm.