Fig.Figure 3(A) Frequency of mitotic phenotypes observed in indicated cell lines with or without DIA treatment (3 repeats, n>50 per repeat, error-bars indicate s. d.). DIA was added for four hours in asynchronous cells followed by 12 hours of imaging. (B) Mitotic duration comparing mock-treated and DIA-treated B1dd/B2ko cells. Time of Entry and Exit was defined by chromosome condensation and decondensation (C) Representative images from live-cell imaging of SiR-Tubulin labelled (red) mitotic B1dd/B2ko cells expressing FusionRed-H2B (green), Mis12-GFP (white); time in mins, scale bar = 5µm. Cells were imaged four hours after DIA treatment. (D) Characterisation of mitotic spindle in proTAME/Apcin (P/A) arrested B1dd/B2ko cells with or without DIA treatment For cell synchronisation (P/A synchronisation) we released from 24-hour Thymidine block into DIA- or PBS-containing medium and added (P/A) 10 hours after release. APC/C inhibition lasted for two to four hours and was followed by fixation and immunofluorescence analysis. (E) Intensities of anti-(pSP) antibody staining in cells treated as in (D) (3 repeats, n>50 per repeat). Key to legend: I-DIA = control interphase cells, M-DIA, M+DIA = control and DIA treated mitotic cells. (F) Cyclin A2 levels were assessed by immunoblotting after 18 hours in 100ng/ml Nocodazole and/or 5µM STLC. 6µM proTAME was added to the indicated samples for the final six hours. (G) Upper panel, confirmation of Cdc27 depletion by immunoblotting 72 hours after siRNA transfection in B1dd/B2ko cells. Lower panel, following 40-hour siRNA transfection B1dd/B2ko cells were treated for 24 hours with Thymidine, released for 10 hours and treated with proTAME and Apcin for additional four hours. At this point, the cells were fixed and stained with tubulin and pericentrin for immunofluorescence analysis. (H) Representative images of mitotic spindles in control- and DIA-treated B1dd/B2ko cells following arrest in mitosis by proTAME/Apcin treatment Tubulin staining is shown in white, pericentrin in green and DAPI in red, scale bar = 5µm. (I) Confirmation of cyclin B1 and B2 depletion 48 hours after siRNA (pool of four different siRNAs) depletion by immunoblotting in WT RPE-1 cells. Asterisks indicate non-specific bands that are not targeted by siRNAs. (J) Additional siRNA depletion of CycB1 and B2 in Ctr and DIA treated B1dd/B2ko cells does not prevent mitotic entry. Cells were released from a 24-hour Thymidine arrest for 10 hours at which point proTAME/Apcin were added for an additional 6 hours before fixation, Hoechst staining and mitotic index scoring. DIA treated mitotic cells. (K) Additional siRNA depletion of CycB1 and B2 in Ctr and DIA treated B1dd/B2ko cells does not change the mitotic phenotypes caused by DIA depletion. and stained with tubulin and pericentrin for immunofluorescence analysis. (L) Immunofluorescence images from mitotic Ctr and DIA treated B1dd/B2ko cells after P/A synchronisation stained with anti-alpha-tubulin (green), anti-gamma-tubulin (red) antibodies and DAPI (blue), scale bar = 5µm. Cells were exposed to ice-cold medium and either fixed immediately after cold exposure, or incubated for 5,10, or 20 minutes at 37° C before fixation.