Fig. 3: Development and characterization of d-astrocytes.

A Representative immunofluorescence images of d-astrocytes from control and patient PKAN_{[Gly420Valfs*30]}a differentiated for 48 days. Astrocytes were stained with the specific markers glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 2 (EAAT2). Nuclei were stained with Hoechst. Scale bars 20 μm. Histograms show the percentage of cells positive for the indicated markers. All data are presented as the mean + SD, n = 6. B Western blot of soluble d-astrocyte homogenates from controls and PKAN patients probed with EAAT2 antibody. Data are representative of three independent experiments. The histogram shows the quantification of EAAT2 normalized to actin by densitometry, n = 3. C OCR measurements of controls and PKAN d-astrocytes analyzed individually. The plot shows OCR normalization to cell number. OCR was measured under basal conditions and after the addition of oligomycin, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), and rotenone/antimycin A (Rot/Ant) for measurement of, other than basal respiration, ATP-linked respiration, maximal respiratory capacity, and nonmitochondrial respiration, respectively. Bars indicate means + SD, n = 3 (two-way ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. D Histogram shows the cell viability obtained by MTT assay. Bars indicate means + SD, n = 3 (one-way ANOVA). **p < 0.01, ***p < 0.001. E Representative immunofluorescence images of d-astrocytes from control and PKAN patients stained with the cytoskeletal marker β-tubulin. Nuclei were stained with DAPI (upper panels). In lower panels, the same images are represented as binary threshold images to better visualize cell borders. Scale bars 40μm. The histogram indicates the major/minor-axis (Ma/mi) ratio of individual d-astrocytes. Bars indicate means + SD (one-way Anova). *p < 0.05, ***p < 0.001.