Fig. 6: Delayed mitotic progression and chromosome mis-segregation in MEF cells expressing MSR-dCas9-Activator.

a Western blot analysis for detection of induced MSR-dCas9-Control, MSR-dCas9-Repressor and MSR-dCas9-Activator in unsynchronised and in RO-3306 synchronised MEF cells. Following release from RO-3306 inhibition, cell samples were collected every 30 min for an interval of up to 2.5 h. Mitotic progression was verified by probing for histone H3S10 phosphorylation (H3S10ph). GAPDH and H3 are shown as loading controls. b Profiles for mitotic progression of RO-3306 synchronised MSR-dCas9-Control, MSR-dCas9-Repressor and MSR-dCas9-Activator MEF cells without doxycycline (-dox, black circles) or with doxycycline ( + dox, red circles) induction. Following release from RO-3306 inhibition, cell samples were collected every 30 min for an interval of up to 2.5 h. Percentages of mitotic cells were quantified by immunofluorescence for H3S10 phosphorylation. For each sample n ≥ 60 cells were analysed. c Confocal imaging of chromosomes in metaphase, anaphase or telophase stages from MSR-dCas9-Control (left panel), MSR-dCas9-Repressor (middle panel) and MSR-dCas9-Activator (right panel) MEF cells without doxycycline (-dox) or with doxycycline ( + dox) induction. Chromosomes were counterstained with DAPI. For each mitotic stage, two representative images are shown. Scale bar is 10 μm. Yellow arrows indicate lagging chromosomes, chromosome bridges, multiple mitotic spindles and non-segregated chromosome sets in MEF cells expressing MSR-dCas9-Activator. Quantification of the mitotic defects is shown in Supplementary Fig. S7.