Figure 9.

Unfolded protein-response genes are upregulated, and increased levels of p-eIF2α protein is found in the Manf^-/- NSCs during differentiation. A-D, qPCR analysis of UPR genes Atf4, Grp78, Chop, Xbp1s, Xbp1t in WT and Manf^-/- NSCs (A), and differentiated cells at DIV1 (B), DIV4 (C), and DIV 8 (D), n = 8-11, *p < 0.05, **p < 0.01, Student’s t test. E, Western blotting from NSCs and differentiated cells at DIV8. F, Quantified intensities of Western blotting bands of p-eIF2α was compared to total amount of (t)eIF2α and teIF2α to intensities of α-tubulin, n = 4, *p < 0.05, Student’s t test. G, TUNEL infrared fluorescence intensities were similar from cultures obtained from Manf^-/- and WT differentiated cells at DIV4 and DIV8. H, Representative photomicrographs of differentiated cells stained for TUNEL (green), DCX (red), and nuclei were counterstained with DAPI (blue); scale bar: 50 µM. I, Differentiated NSCs at DIV8 death assessed by TUNEL staining. J, WT and Manf^-/- NSCs at DIV1, DIV2, and DIV8 incubated in HPG. Diagonal arrows denote the cytoplasmic localization of synthesized proteins and arrowhead indicates newly synthesized proteins spread to growing neurites. K, Quantified ratio of Click-iT HPG fluorescence to DAPI fluorescence intensities in WT and Manf^-/- cells, two-way ANOVA, p < 0.01, Bonferroni’s post hoc test, ***p < 0.001.