Fig. 8. Simultaneous depletion of DmSMC4 and DRAD21 in Drosophila S2 cells. (A) Protein extracts from different concentrations of cells were immunoblotted with anti-DRAD21 antibody to determine the level of detection of the antibody. (B) Protein extracts from control or DRAD21-depleted cells were immunoblotted to detect DRAD21, DmSMC4 and -tubulin as a loading control. Note that significant depletion of DRAD21 is obtained 72 hours after treatment with RNAi while the levels of DmSMC4 remain unchanged. Cells were also treated with dsRNA for DRAD21 and DmSMC4 simultaneously and protein extracts prepared at different times and immunoblotted to reveal DRAD21, DmSMC4 and -tubulin as loading control. Significant depletion of both proteins was obtained after 96 hours of treatment therefore all phenotypic and quantification analysis was carried out at this time. (C) Cells depleted of DRAD21, stained for DRAD21 and DNA, show premature sister chromatid separation and highly condensed chromatids. (D) Cells depleted of both DmSMC4 and DRAD21, stained for DRAD21, DmSMC4 and DNA, show abnormal chromosome condensation and poorly defined sister chromatids during prometaphase and during anaphases have extensive chromatin bridges. (E) Quantification of mitotic index of control cells and cultures treated with different dsRNAs shows some accumulation of mitotic cells after depletion of DRAD21 or simultaneous depletion of DRAD21 and DmSMC4. (F) Quantification of mitotic progression after treatment of cells with different dsRNAs. Depletion of DmSMC4 does not affect mitotic progression while depletion of DRAD21 causes a significant prometaphase arrest with few cells in metaphase or anaphase. Simultaneous depletion of DmSMC4 and DRAD21 causes a significant accumulation of cells in anaphase and telophase. The number of cells used for the quantifications shown in E and F was: control n=1148, DmSMC4 RNAi n=1038, DRAD21 RNAi n=1923 and simultaneous DmSMC4/DRAD21 RNAi (double) n=1801. Scale bars: 5 µm.