Fig. 1. Depletion of DmSMC4 in Drosophila S2 cells by dsRNAi. (A) Depletion of DmSMC4 was monitored over time by western blot analysis. Total protein extracts from 10⁶ cells were separated by SDS-PAGE and immunoblotted with anti-DmSMC4 or -tubulin as a loading control. Quantification of depletion was determined by normalising for -tubulin. (B) Protein extracts of different concentration of control cells were immunoblotted with anti-DmSMC4 antibody to determine the level of antibody detection. (C) Viability index of control and DmSMC4 dsRNAi-treated cells was determined by Trypan Blue staining. (D) Proliferation of control or DmSMC4 dsRNAi-treated S2 cells was monitored throughout the experiment. (E) Quantification of the mitotic index during the course of the experiment in control cells and cells treated with DmSMC4 dsRNAi was determined by counting phosphohistone H3-positive cells. (F) Anti-phosphohistone H3-positive cells were also used to quantify mitotic progression in control (C) and RNAi (R)-treated cells. Numbers of cells counted for D and E is shown in Table 1. (G) Phenotypic analysis of phosphohistone H3 positive cells in control and DmSMC4 dsRNAi-treated cells. After depletion of DmSMC4, phosphohistone H3 staining persists on chromatin bridges during telophase (arrow). Representative images of metaphase chromosomes at a higher magnification are shown in the insets. Note that sister chromatids do not resolve after DmSMC4 depletion. Scale bars: 5 µm.