Fibronectin and collagen

Dorsal skin was harvested from euthanized from Flii^+/−, WT and Flii^Tg/Tg mice skin (age 8–10 weeks) and sterilized in iodine 10% antiseptic saline solution. Skin was cut into 1 cm wide squares and digested in serum-free DMEM (Sigma, Melbourne, Australia) buffered Dispase II (0.05 g/mL, Roche Diagnostics, Sydney, Australia) solution overnight at 4 °C, followed by removal of dermis the next day. The epidermal tissue was then digested in 0.25% trypsin/HBSS (Sigma) for 20 min at 37 °C, followed by addition of FBS (1:10) and filtering through a 70 μm and 40 μm nylon strainer (BD Biosciences, North Ryde, Australia). Isolated cells were plated into T75 flasks pre-coated with fibronectin and collagen (50 μg/mL, Invitrogen, Mount Waverley, Australia) containing low calcium Epilife medium (10% FBS in Epilife, Thermofisher, New South Wales, Australia) and cultured for 2 weeks until next passage.

MET-1 cells +/− Flii siRNA were plated on glass coverslips pre-coated with fibronectin and collagen (50 μg/mL, Invitrogen) in the wells of a 6-well plate at a density of 20,000 cells in Epilife medium containing 10 μM BrdU (Sigma-Aldrich, Sydney, Australia) [34 (link)]. BrdU was allowed to incorporate into the DNA synthesis (pulse-process) for 24 h followed by washing in sterile PBS twice. New Epilife medium containing 5 μM cytochalasin D (cytD) was then added to the cells to arrest cytokinesis (chase-process) for 24 h, followed by washing in sterile PBS twice. The cells were subsequently washed twice in washing buffer (0.5% BSA in PBS) and fixed in 4% paraformaldehyde. Fixed cells were permeabilized in buffer (0.2% Triton X-100 in washing buffer) followed by blocking in washing buffer. Primary antibodies including BrdU (1:400, Sigma-Aldrich, Sydney, Australia) and pH3(s28) (1:400, Abcam, Sydney, Australia) were diluted in washing buffer and applied. Species-specific Alexa Fluor 568 or 633-conjugated secondary antibodies (1:400, Invitrogen, Melbourne, Australia) were diluted in washing buffer and applied along with Oregon Green 488 Phalloidin (1:400, Thermo Fisher Scientific, Melbourne, Australia) and DAPI (1:5000). Stained cells were imaged and positive cells counted using Olympus cellSens Dimension software.