Figure 4. Tissue confinement determines cell growth rate and signaling.

(A and B) Images of Cell Trace (A) and YAP (B) at $\mathit{\text{Δ}}t=48$ h in expanding colonies with initial size, ${\text{A}}_0$, varying from 0.8 to 7.1 mm², subconfluent colonies and confluent conditions. In (A), overlay shows initial colony size ${\text{A}}_0$ (dashed circle) and expansion $v\mathit{\text{Δ}}t$ (arrow). Note different scale bars in (A) and (B).

(C) CellTrace images of expanding monolayers with ${\text{A}}_0=0.8$ mm at $\mathit{\text{Δ}}t=48$ h in the presence and absence of 500 nM PND1186 (+FAKi) to illustrate the effect of changes in $v\mathit{\text{Δ}}t$.

(D) Quantification of cell growth and YAP signaling at varying tissue confinements with $\mathit{\text{Δ}}t=48$ h across multiple experiments. Each data point is an average of multiple colonies from one experiment (see STAR Methods). YAP activity is determined by the nuclear-to-cytoplasmic ratio of anti-YAP intensity, quantified for >150 cells in each experiment. Growth is plotted against the time averaged confinement and YAP activity is plotted against the final confinement. Data are from 6 independent experiments (cell growth) and 3 independent experiments (YAP activity).

(E) MDCK FUCCI cells in expanding colony with ${\text{A}}_0=1.8$ mm² and $\mathit{\text{Δ}}t=12$ h.

(F) Quantification of cell growth and fraction of cells in the S/G2/M cell-cycle states as a function of the average tissue confinement. Each data point is quantified from >3 colonies in a single experiment with ${\text{A}}_0=0.8$, 1.8, or 7.1 mm² and $\mathit{\text{Δ}}t=12$ h. Data are from 2 independent experiments. See also Figure S11.