Transduction of FITC-labeled WT and K7A/K9Q chimeric ZHHH, human, and zebrafish Ngb proteins into PC12 cells. About 3 ?m of FITC-labeled (green) Ngb protein was applied to PC12 cells in the presence of FM4-64 (red), a fluorescent marker of endocytosis. The cells were then incubated for 24 h under normoxic conditions. The living, unfixed cells were observed directly by confocal fluorescence microscopy. Scale bar, 10 ?m.

Effects of NGF, WT chimeric ZHHH Ngb, human Ngb, zebrafish Ngb, K7A/K9Q chimeric ZHHH Ngb double mutant, and H67V, E56Q, E63Q, or E121Q chimeric ZHHH Ngb single mutants on neurite outgrowth of PC12 cells after 5 days of incubation. (A) Differential interference contrast images of PC12 cells incubated with PBS, NGF (100 ng·mL^?1), or Ngb (10 ?m) for 5 days. Scale bar, 10 ?m. (B) The percentage of cells with processes longer than their cell body after 5 days of incubation. All data are expressed as means ± SEM from three independent experiments, each performed in triplicate. Data were analyzed by one-way ANOVA followed by Tukey?Kramer post hoc tests. *P < 0.05, **P < 0.01 compared with PBS. ^##P < 0.01 compared with WT chimeric ZHHH Ngb.