Page 156 - scheppingen

six
154
Figure 2. miR147b decreases inflammation in astrocytes. Quantitative real-time PCR of miRNA or mRNA expression (A-C, E) and ELISA analysis of protein levels (D) in fetal human astrocyte cultures. A Astrocytes were treated with IL-1β (10 ng/ml) or LPS (100 ng/ml) for 1, 6, 24, 30 and 48 hours. miR147b was up-regulated after stimulation with IL-1β with peak expression at 30 hours. B TPCA-1 decreased both miR146a and miR147b expression. C-E Astrocytes were transfected for 24 hours with miR147b mimic (50 nM) followed by 24 hours of IL-1β stimulation. miR147b mimic decreased the mRNA levels of pro-inflammatory markers IL-6 and COX-2 and reactivity marker C3 (C), and the level of IL-6 in culture supernatants (D). E miR147b mimic decreased the mRNA levels of predicted targets DEPTOR and ADAM15. Experiments were performed in triplicate and data are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 compared to control, Kruskall-Wallis with Dunn’s post-hoc test (A) and Mann Whitney U test (B-E).
Altered neural stem cell differentiation after miRNA transfection
NSCs were grown in floating conditions and secondary cultures could be generated after dissociation (Fig 4 A and B). NSCs were positive for stem cell markers Nestin and SOX-2 and gained positivity for neuronal or astrocytic markers (βIII-tubulin and GFAP, respectively) after differentiation for 7 or 14 days (Fig 4 C-O).
The effects of miR146a and miR147b on neuronal versus astroglial cell fate dif-