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. 2017 Jan 6;7:39985. doi: 10.1038/srep39985

Figure 7. NT5C regulates cell motility and cell spreading.

Figure 7

(a) NT5C regulates single cell motility. (left) HT1080 cells stably expressing siRNA were seeded into 96-well plates. Images were acquired every 10 min for 80 time points. Distance travelled is expressed relative to siControl cells. The experiment was repeated 3 times independently. n = 235–301 cells per cell line; Error bars are sem; ***p < 0.001. (right) Representative immunoblot from experimental cells. (b) NT5C expression rescues motility defect. HT1080 cells stably expressing siRNA and Flag-NT5C rescue constructs were processed as in (a). Analysis of distance travelled is expressed relative to cells expressing Control siRNA and empty vector cDNA. The experiment was repeated 4 times independently. n = 138–472 cells per cell line. Error bars are sem; *p < 0.05, **p < 0.01. (c) NT5C regulates single cell spreading. HT1080 cells stably expressing siRNA were trypsinized, seeded onto fibronectin coated plates and fixed at 45 min post-seeding. (left) Cell area was determined manually in ImageJ and is expressed relative to siControl cells. The experiment was repeated 3 times independently. n = 326–431 cells per cell line; Error bars are sem; *p < 0.05, **p < 0.01. (top right) Representative image. (bottom right) Representative immunoblot from experimental cells. (d) NT5C expression rescues spreading defect. (top) HT1080 cells stably expressing siRNA and Flag-NT5C rescue constructs were processed as in (c). Cell spreading is expressed relative to cells expressing Control siRNA and empty vector cDNA. The experiment was repeated 4 times independently. n = 555–659 cells per cell line; Error bars are sem; *p < 0.05, **p < 0.01, ***p < 0.001. (bottom) Representative immunoblot from experimental cells. (e) NT5C knockdown impairs cell spreading in primary MEFs. Primary MEFs stably expressing siRNA were processed as in (c) 96 h after 4-OHT treatment. (left) Cell spreading is expressed relative to Pik3caWT/WT cells expressing Control siRNA. The experiment was repeated 3 times independently. n = 244–356 cells per cell line; Error bars are sem; **p < 0.01. (right) Representative immunoblot from experimental cells.