Figure 4. WEE1 inhibition in FANCM- or BRIP1-depleted cells causes replication stress and unscheduled mitosis A.

Immunofluorescence images showing induction of pan-nuclear γH2AX (green) and pH3 (red) staining in WiDr cells transfected with siCON1, siFANCM or siBRIP1 for 48 hours, and exposed to 250 nM MK-1775 for the indicated times. DNA was counterstained with DAPI (blue). Unscheduled mitosis is characterised by double γH2AX⁺pH3⁺ staining (yellow, quantified in Supplementary Fig. 4B).

B. Representative flow cytometry plots measuring pH3 (mitosis) and γH2AX (in green; events were back-gated from γH2AX versus DNA content plots shown in Supplementary Fig. 4D) in cells transfected with siCON1, siFANCM and siBRIP1 and exposed to 250 nM MK-1775 for the indicated times. Normal mitotic pH3⁺ cells are shown in red. Arrows indicate premature mitotic cells that are γH2AX^highpH3⁺ double positive. A back-gated population of S-phase cells that is γH2AX^low is shown in green. 2N DNA content indicates cells in G1 phase, 4N DNA content indicates cells in either G2 or M phase.

C. Quantification of mitosis (pH3⁺) and premature mitosis (γH2AX^highpH3⁺) by flow cytometry analysis of cells treated as described in B. For γH2AX^highpH3⁺ cells, * denotes P<0.05 versus siCON1 at 16h (ratio paired Student’s t-test).

D. Western blots of FANCD2 in cells exposed to 300, 600 and 1000 nM of MK-1775 for 4 or 8 hours (left) or to 250 nM MK-1775 for indicated times (right). Hydroxyurea (HU) or mitomycin C (MMC) treatment for 24 hours served as positive controls. Ezrin and β-Tubulin were used as loading controls. S, short non-ubiquitinated FANCD2 isoform. L mono-ubiquitinated long isoform.