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. 2017 Sep 29;4(5):ENEURO.0214-17.2017. doi: 10.1523/ENEURO.0214-17.2017

Figure 3.

Figure 3.

Effects of MANF on neuronal differentiation. A, Time line of the differentiation of NSCs. NSC expansion was done for 1 d in floating conditions. At day 1, differentiation begun without EGF and FGF-2 for 7 d in adhering conditions. B, At DIV1, DIV2, and DIV4, differentiated cells were stained with antibodies against neuronal markers TuJ1; scale bar; 50 µm. C, Neurite length were measured from TuJ1-positive cells. Manf-/- cells displayed significantly shorter neurite length. Exogenous rhMANF induced neurite outgrowth at DIV2 but not at DIV4 of Manf-/- cells, two-way ANOVA, post hoc Bonferroni test; **p < 0.01, ***p < 0.001; scale bar: 50 µM. D, E, Representative photomicrographs of differentiated cells double-stained for TuJ1 (green), DCX (red), MAP2 (green), and GFAP (red), and nuclei stained with DAPI (blue); scale bar: 50 µM. F, Ratios of GFAP-, DCX-, TuJ1-, and MAP2-positive cells in relation to DAPI+ nuclei (n = 9). G, H, At DIV8, neurite extension were measured from TuJ1-positive cells. Manf-/- cells displayed significantly shorter processes than WT cells (n = 9); scale bar: 20 µM. I, Lysates from WT and Manf-/- cells after differentiation were immunoblotted with MAP2, TuJ1, GFAP, DCX, and GAPDH antibodies. J, Protein levels were quantified in relation to levels of GAPDH, a housekeeping protein, n = 9; *p < 0.05, **p < 0.01, Student’s t test.