Figure 2

Protective effect of Ntn-1 on hypoxia-induced UCB-MSC apoptosis via DCC and IN α6β4. (a) Total RNA from UCB-MSCs was reverse transcribed, and DCC, UNC5A, UNC5B, UNC5C, and neogenin cDNA were amplified by PCR, as described in Materials and Methods. Each example shown is representative of five independent experiments. (b) Cells were pretreated with Ntn-1 (10 ng/ml) for 30 min before being exposed to hypoxia for 72 h, and DCC, IN α6, and IN β4 were detected by western blot. (c) Cells were incubated in the presence of Ntn-1 for 24 h and then harvested. Cell lysates were analyzed by western blotting with antibodies to INs α6 and β4. Anti-DCC immunoprecipitation was analyzed by western blotting with INs α6 and β4 antibodies. (d) Control lysate was subjected to discontinuous sucrose density-gradient fractionation, after which DCC, IN α6, IN β4, caveolin (Cav)-1, Cav-2, flotillin-2, and β-actin were detected. Each fraction was assessed by western blot. (e and f) Cells were pretreated with DCC function-blocking antibody (2.5 μl/ml) or combination of INs α6 and β4 function-blocking antibodies (2.5 μl/ml) for 30 min prior to hypoxia with Ntn-1 exposure for 72 h; Bax and Bcl-2 were detected by western blot. (b–f) Each example shown is representative of five experiments. The lower or right part (b, e and f) depicting the bars denotes the mean±S.E. of five independent experiments for each condition determined from densitometry relative to β-actin. *P<0.05 versus control, **P<0.05 versus hypoxia alone. ROD, relative optical density