Nuclear / Cytoplasmic (N/C) ratio analysis

The nuclear/cytoplasmic ratio was quantified by measuring the mean fluorescence intensity of a nuclear region (Inucleus) and the intensity of a cytoplasmic region immediately adjacent (Icytoplasm). The nuclear region was determined from the Hoechst staining. The ratio was calculated using the following formula:

Where Ibackground is the mean fluorescence intensity of a region outside of the cell. For the quantification of the mechano-reporter L_NLS-41-GFP with time, the N/C ratio was calculated at each time point. For all drug treatment experiments, the N/C ratio at each timepoint was normalized to the N/C ratio prior to the addition of the compound. For quantification of the N/C mechano-reporter L_NLS-41-GFP ratio during in-situ gel softening experiments, a single confocal plane was selected and the N/C ratio was normalized between the pre-softened N/C ratio and the ratio at the final timepoint.