Fig. 6
From: Full UPF3B function is critical for neuronal differentiation of neural stem cells
A functional NMD pathway is important for neuronal differentiation. a. Treatment with NMD inhibitor Amlexanox affects neuronal differentiation of neural stem cells. Neural stem cells were differentiated in the presence of DMSO or 10 μM Amlexanox or without any further addition. Neurons were analysed 3 days and 6 days after induction of differentiation as described. Shown are representative pictures of neurons with tracing, and tracing of 2 additional neurons after 6 days of differentiation illustrating a reduction in branching in Amlexanox treated neurons. The scale bar represents 20 μm. Results are plotted showing the median, with boxes spanning 25th- 75th percentiles and the crossbars indicating the 10th and 90th percentiles. Experiments were done independently 3 times with similar outcomes and the data were combined for presentation. Numbers of cells analysed are indicated. The Asterisk indicates that branching of Amlexanox treated neurons is significantly reduced compared to neurons differentiated without addition any addition or DMSO only (Kruskal Wallis test followed by Dunn's multiple comparison test; P < 0.05). b. Expression of mutant UPF3B proteins that affect neuronal differentiation causes an increase in mRNA levels of selected NMD substrates. Neural stem cells were transduced with lentivirus without insert (control) or expressing either UPF3B, UPF3B-Asp160 or UPF3B-His366 protein. 24 h after transduction, differentiation was induced. RNA was isolated after six days of differentiation and gene expression was analysed by reverse transcription followed by quantitative PCR. mRNA levels of NMD substrates Atf4 and Arhgap24 isoform 1, and of transcripts encoding all shorter Arhgap24 isoforms not subject to NMD control were measured and standardised using Gapdh mRNA as reference. Atf4 and Arhgap24 mRNA levels in cells transduced with the control virus were set as 1. Asterisks indicate values significantly increased compared to levels in cells transduced with the control virus (one-way ANOVA followed by Dunnett's test; P < 0.05)