A-431-S cells were seeded into a 24-well plate and grown to confluence. Each monolayer was subjected to a 45 min calcium-free pulse and calcium was restored for 2, 4, 6, or 8 h, as described above. The no-pulse control was maintained in normal calcium throughout the experiment, while calcium was not restored for the time 0 control. After the calcium-free pulse assay, 1 U/mL Dispase II (Sigma-Aldrich) was added to each well for approximately 30 min – until the monolayer was entirely lifted from the plate. A P1000 tip was clipped and lubricated with FBS before subjecting each cell sheet to mechanical stress using a uniform number of pipette aspirations. The number of aspirations for each independent experiment was determined by the number required to produce countable fragments in the no-pulse control. After fragmentation, the samples were fixed with 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) and fragments were manually counted under a dissection microscope. The assay was performed independently three times, with each experiment comprising three technical replicates per time point.
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