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. Author manuscript; available in PMC: 2019 Apr 26.
Published in final edited form as: Mol Cancer Ther. 2015 Feb 11;14(4):865–876. doi: 10.1158/1535-7163.MCT-14-0845

Figure 3. WEE1 inhibition promotes unscheduled mitosis after CHK1 and MYT1 depletion A.

Figure 3

Clonogenic survival of WiDr cells transfected with siCON1, siCHK1 or siMYT1, with colonies counted after 10 days of MK-1775 exposure. The number of colonies was normalised to untreated controls for each siRNA separately (clonogenic survival before normalisation in the absence of MK-1775: siCON1: 69%, siMYT1: 67% and siCHK1: 35%). Knockdown of CHK1 and MYT1 was confirmed by Western blot (β-Actin, loading control).

B. Flow cytometry analysis of γH2AX and pH3 after transfection with siCON1, siCHK1 and siMYT1. Cells were exposed to 200 nM MK-1775 for the indicated times. γH2AXhighpH3+ cells represent unscheduled mitosis. Error bars represent SEM of two independent experiments.

C. Immunofluorescence images showing induction of pan-nuclear γH2AX (green) and pH3 (red) staining in WiDr cells transfected with siCON1, siCHK1 or siMYT1 and exposed to 300 or 600 nM MK-1775 for 8 hours. Double γH2AX+pH3+ staining (yellow) indicates premature mitosis. DNA was counterstained with DAPI (blue).

D. WiDr cells transfected with siCON1 (black bars) or siMYT1 (grey bars) were exposed to 250 nM MK-1775 for the indicated times. Cells were stained for γH2AX and imaged by confocal microscopy. Representative immunofluorescence images show premature mitotic cells (indicated by arrows) after 24 hours of MK-1775 exposure. Bar charts show number of γH2AX-positive cells (left), γH2AX intensity (middle) and premature mitosis (right) in siMYT1-transfected cells compared to siCON1. Error bars are SEM of two independent experiments.