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Migratory ability of NPC S18 cells is suppressed by knocking down SPINK6 expression. S18-NC and S18-SPINK6-KD cells (GFP-tagged) were seeded in a 96-well plate (2000 cells per well) and cultured in DMEM with 10% FBS in 5% CO2 at 37 °C for 24 h. Next, the plate was placed under a Nikon ECLIPSE Ti2 Inverted Microscope System supplied with 5% CO2 at 37 °C. Images of random fields at 100× magnification were taken automatically every 30 min for 72 h. A total of 145 sequential images of each field were taken, generating a video to display the movement of the cells at a speed of 15 FPS (frame/sec) (see Videos 1 and 2). The locations and moving tracks of the cells were analyzed using Image J software and the distance of movement between sequential images every 30 min were calculated. The videos displaying the movement of S18-NC and S18-SPINK6-KD cells are shown in Videos 1 and 2. The accumulated distance traveled in 72 h of 100 randomly selected cells from each cell line were calculated and are shown in A and B.

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