Figure 4.

Ras Activation Promotes Mitotic Rounding under Confinement

(A) Schematic of cells dividing in confinement under compliant (~5 kPa) and stiff (~ 30 kPa) polyacrylamide gels.

(B) Plot of maximum metaphase length versus height for unconfined MCF10A cells and cells under soft (~5 kPa) and stiff (~ 30 kPa) gels. Measurements were taken from confocal time-lapse images as shown in (C). Metaphase was taken as the frame before anaphase.

(C) XY and XZ confocal slices of metaphase MCF10A+hRas^v12 cells, MCF10A cells, and MCF10A cells treated for 5–10 h with 10 μM selumetinib. Cells were pre-treated with 100 nM SiR-DNA and SiR-actin to label DNA and actin. Scale bars represent 20 μm.

(D) Boxplot showing metaphase cell length for MCF10A+hRas^v12, control MCF10A, and MCF10A + 2 μM PD 184352 without confinement and under soft and stiff gels. Data come from time-lapse movies for 15 h after confinement onset. The MEKi was added immediately before confinement and mitoses were analyzed 5–15 h post addition and confinement. p Values calculated using Mann-Whitney ^∗∗∗p < 0.001.

(E) Graphs showing cell area for control MCF10A cells and MCF10A+hRas^v12 entering mitosis under soft and stiff gels, aligned so that t = 0 is NEB.