Figure 3
Co-depletion of Deco and Naa50/San significantly enhances Drosophila S2 cells chromosome segregation defects.
All cells in this figure were analyzed 72 hours after RNAi-treatment. (A–D) Selected frames from time-lapse videos (see Movies S7,S8,S9,S10) of control RNAi, san RNAi, deco RNAi, and san RNAi deco RNAi co-treated S2 cells (arrowheads indicate single chromatids). san RNAi-treated and deco RNAi-treated cells showed chromosome segregation defects during anaphase (B,C,F; see arrowheads in B and C) and a significant arrest during mitosis (E), however detectable levels of cells with single chromatids during metaphase were only detected in san RNAi-treated cells (F). Co-depletion of Naa50/San and Deco significantly enhanced the mitosis arrest (E) and frequency of cells with single chromatids (D,F; see arrowheads in D). (E) Mitotic index (% of phospho-H3 (pSer10) positive cells) for control RNAi, san RNAi, deco RNAi, and san RNAi deco RNAi co-treated cells (72 hours after RNAi-treatment) was, respectively, 2.4% ± 0.2 (n = 1928), 7.1% ± 0.7 (n = 1056), 5.0% ± 1.0 (n = 1469), and 11.5% ± 1.4 (n = 965). The following mitotic index differences are statistically significant for control RNAi comparing to san RNAi treated cells or to san RNAi deco RNAi co-treated cells; and for san RNAi treated cells comparing to san RNAi deco RNAi co-treated cells (p < 0.05 Student’s t-test). (F) Frequency of cells with chromosome segregation defects during anaphase after control RNAi, san RNAi, deco RNAi, and san RNAi deco RNAi co-treated cells (72 hours after RNAi-treatment) was, respectively, none (0%) (n = 7), 18% ± 0.1 (n = 17), 20% ± 0.1 (n = 16), and 18% ± 0.1 (n = 20). (F) Frequency of cells with single chromatids after control RNAi, san RNAi, deco RNAi, and san RNAi deco RNAi co-treated cells (72 hours after RNAi-treatment) was, respectively, none (0%) (n = 7), 5.6% ± 0.1 (n = 17), none (0%) (n = 16), and 66.8% ± 0.2 (n = 20). The difference between frequency of cells with single chromatids in san RNAi treated cells and san RNAi deco RNAi co-treated cells is statistically significant (p < 0.01 Student’s t-test). Drosophila S2 cells stably expressed GFP-Histone H2B (green) and α-Tubulin-mCherry (red) (A–D)70. All images were obtained using maximum intensity projections of Z-stacks (0.8 μm stacks of 5 sections each). Scale bars equal 10 μm.
