Figure 6.

Neurite growth features of CLN1 transfected cell lines following neuronal differentiation in RA-NBM medium. (A) Outgrowth of neurites in CLN1 transfected cells lines were evaluated by β-III tubulin immunostaining during neuronal differentiation. SH-p.wtCLN1 cells showed less arborized, stunted neurites in comparison to mock-transfected cells and other CLN1 transfected cell lines. Nuclei are marked in blue; DIV, days in vitro; scale bars equal to 50 μm. For a qualitative evaluation of cell morphology see also Figure S6 in the supplementary Materials. (B) The morphometric analysis of β-III tubulin immunolabeled processes pinpointed a significant paucity of neurites longer than 30 μm in SH-p.wtCLN1 cells, in accordance with the bioinformatic findings. Likewise, a similar, even though not significant, trend was seen for SH-p.L222P clone. Higher variability was detected in SH-p.M57Nfs*45. These findings resembled the results about neurofilament immunolabeled structures which are described in Figure S7 of supplementary Materials. Mean ± SEM of three independent experiments; One-way ANOVA followed by Bonferroni’s post-test; *p < 0.05. (C) Immunoblotting analysis of β-III tubulin demonstrated a reduced expression of this cytoskeletal marker in SH-p.wtCLN1 only, in accordance with the decreased number of neurites. Arrow pinpoints to a decreased expression of β-III tubulin in SH-p.wtCN1 cells. (D) Semi-quantitative WB analysis confirmed a decreased expression of β-III tubulin in SH-p.wtCLN1, following differentiation in RA-NBM medium. GAPDH served as internal standard; a.u. arbitrary units; mean ± SEM of three independent experiments; unpaired t-test; *p < 0.05.