Dispase dissociation assays.

Rheb WT and cKO cells were grown on uncoated 6-well plates. Confluent keratinocyte monolayers were pretreated with inhibitors for 24 hours prior to switch in 1.2 mM calcium for 18 hours. At assay, cells were rinsed with PBS and incubated in 2.5 U/ml dispase solution (Sigma-Aldrich) in PBS for 15 minutes at 37°C. Detached sheets were transferred to 15-ml conical tubes in PBS and stressed by mechanical rotations. Resulting fragments were visualized and images captured.