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. 2014 Apr 16;23(16):1870–1882. doi: 10.1089/scd.2013.0632

FIG. 1.

FIG. 1.

Effects of Ntn-1 on human UCB-MSC migration. (A) Dose response of Ntn-1 in Oris™ cell migration assay. Cells were treated with different doses of Ntn-1 (0.01–100 ng/mL) for 24 h. Fluorescence in the analytical zone was quantified with a plate reader. Data represent mean±SE of five independent experiments with triplicate dishes. *P<0.05 versus vehicle. (B) Time response of Ntn-1 in Oris cell migration assay. Cells were treated with 50 ng/mL Ntn-1 for various times (0–48 h). *P<0.05 versus 0 h. (C) Cells treated with 50 ng/mL Ntn-1 for 24 h were visualized by calcein AM staining using Oris cell migration assay. n=5. Scale bars represent 200 μm (magnification,×40). (D) Cells were pretreated with DCC blocking antibody (2.5 μL/mL) or combination of INα6 and INβ4-blocking antibodies (2.5 μL/mL) for 30 min prior to Ntn-1 exposure for 24 h. Oris cell migration assay was performed and quantified the value of fluorescence. Data represent mean±SE of five independent experiments with triplicate dishes. *P<0.01 versus vehicle. #P<0.01 versus Ntn-1 alone. (E) Wound-healing assay was performed and stained with phalloidin-AlexaFluor 488 to identify the migrating cells. n=5. Scale bars represent 100 μm (magnification,×100). (F) Effect of mitomycin C in Ntn-1-induced cell migration. Cells were pretreated with 1 μg/mL mitomycin C for 30 min prior to 50 ng/mL Ntn-1 exposure for 24 h. Oris cell migration assay was performed and quantified the value of fluorescence. Data represent mean±SE of four independent experiments with triplicate dishes. *P<0.01 versus vehicle. DCC, deleted in colorectal cancer; IN, integrin; Ntn-1, Netrin-1; RFU, relative fluorescence units; SE, standard errors; UCB-MSCs, umbilical cord blood-derived mesenchymal stem cells.