FIGURE 3:

Cell density–dependent Erk1/2 activation drives spatially constrained Erk1/2 activity throughout sheets. (A) Immunofluorescence staining of active Erk1/2 in fixed sheets of HaCaT cells reveals TGFβ-dependent spatially constrained Erk1/2 activation at the leading edge at 24 h post–ligand stimulation. (B) Cellular density was measured with spatial resolution throughout sheets by quantifying the cellular area as a function of distance from the leading edge (position) after 24 h of ligand stimulation. (C) Titrations of cellular density in the presence or absence of TGFβ at 24 h reveal that high cell density inhibits Erk1/2 activity in a ligand-independent manner. (D) HaCaT EKAR-NES cells were used to measure the effect of cellular density on the spatial profile of Erk1/2 activity at 24 h post–ligand stimulation. The displayed heat map key represents the calculated FRET ratio from the EKAR-NES biosensor.