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Fig. 2 | Journal of Neuroinflammation

Fig. 2

From: The smoothened agonist SAG reduces mitochondrial dysfunction and neurotoxicity of frataxin-deficient astrocytes

Fig. 2

Chronic treatment with SAG improves cell metabolism of FXN-deficient HAs. A Schematic representation of SAG chronic treatment of FXN-deficient astrocytes. B mRNA expression levels of SHH signaling pathway members were determined by qPCR. Gene expression was normalized to GAPDH and quantified by the comparative Ct method (n = 4). C The graph on the left shows FXN mRNA transcript levels in HAs in all experimental groups, analyzed by qPCR (n = 4). The top panel and the graph on the right show a representative immunoblot and quantification of FXN levels in HAs after transduction and treatment with SAG (n = 4). D Cellular metabolic activity (MTS reduction) in HAs after SAG chronic treatment (n = 8). E Representative images of live (green) and dead cells (red) estimated by the calcein/PI uptake assay (n = 3). HAs transduced and treated with SAG were incubated with both dyes and analyzed by fluorescence microscopy. Scale bars: 200 μm. The graph on the right shows the quantification of cell death, estimated as the % of PI positive cells per field. F Representative immunoblots and quantification of cleaved-PARP1, p53 and p21 levels for the indicated groups. Same amounts of protein were immunoblotted and probed with the corresponding antibody and β-actin (as loading control) (n = 4). G Representative confocal images (left) and quantification (right) of HAs subjected to the indicated experimental conditions (n = 3). Cells were fixed and stained with specific antibodies against S100β and cleaved Cas3, and with To-Pro-3 to stain the nuclei (blue). Scale bars: 30 μm. In all graphs, data were normalized to the untreated group (data not plotted) and represent mean values ± S.E.M. In B–G data were evaluated by one-way ANOVA followed by Tukey’s post hoc test

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