In vitro Wound-healing Assay

For the wound-healing assay, cells were plated at a several densities ranging from 0.5–1×106 cells/well in a 6-well plate (3 wells per cell line) and cultured for 24 hours under standard culture conditions. The day of the assay, control and knockout cells showing comparable densities were selected for assessment. Three parallel lines per well were ‘drawn’ with gentle pressure, using a sterile 1000 µl pipette tip. Cells were washed twice with PBS to remove cell debris before adding pre-warmed standard culture medium. The plate was then placed into the temperature- and CO2-controlled environmental chamber (OKO lab) of a Nikon Ti Eclipse microscope with perfect focus, motorized stage and Zyla 4.2 sCMOS camera (Andor) driven by a NIS Elements V4.13 software package. Wound closure (1 region of interest per scratch) was recorded by phase contrast using a 10× air objective at a frame rate of 1 image/10 minute for 24 hours. For quantification in ImageJ, the wound area was measured manually in the first and last frame of each dataset (8 per cell line) and results were expressed as efficiency of wound closure (100−[(100/area first frame)*area last frame]).