Fig. 3
The effects of Bit1 depletion on cell proliferation, migration and invasion abilities in ESCC EC9706 and TE1 cells. a and b Bit1-shRNA significantly suppressed cell growth on days 2, 3 and 4 in EC9706 and TE1 cells. EC9706 and TE1 cells at a density of 5,000 cells per well were seeded into 96-well culture plates. When cells were cultured for 24 h, and the pSilencer3.1-H1-neo-Bit1-shRNA and pSilencer3.1-H1-neo-negative-shRNA were utilized to transfect EC9706 and TE1 cells. The number of metabolically active cells was assessed by 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay at each indicated time (1d, 2d, 3d, 4d, 5d, 6d and 7d). c and e Bit1 knockdown markedly impeded cell migration in EC9706 and TE1 cells. 4 × 105 of EC9706 and TE1 cells were seeded into 6-well culture plates. After transfection with pSilencer3.1-H1-neo-Bit1-shRNA and pSilencer3.1-H1-neo-negative-shRNA for 24 h, in vitro scratch wounds were created by scraping the cell monolayers with a 200 μl sterile pipette tip. The wounded cultures were allowed to grow for 36 h at 37 °C. At 12 h, 24 h and 36 h, photomicrographs were taken at the same position to evaluate the alterations of cell migration ability. d and f Cell migration was assessed by measuring migration distances from the wound edges. *P < 0.05, compared with untreated and negative group. g Bit1 downregulation contributed to the decrease of cell invasion ability in EC9706 and TE1 cells. After transfection with pSilencer3.1-H1-neo-Bit1-shRNA and pSilencer3.1-H1-neo-negative-shRNA for 24 h, EC9706 and TE1 cells (3-5 × 104 per well) were seeded to ECM gel pre-coated, porous upper chamber inserts and allowed to grow at 37 °C in a CO2 incubator. The cells that invaded to the bottom surface of the insert were fixed with methanol and stained by 0.5 % crystal violet, and was then subjected to microscopic inspection. h and i The invasive cell numbers were counted to evaluate the effect of Bit1-shRNA on cell invasion ability in EC9706 and TE1 cells, **P < 0.01, compared with untreated and negative groups