Fig. 5

FXN deficiency induces a reactive phenotype in HAs that is attenuated by SAG. In A–D, HAs were cultured under standard conditions (untreated) or transduced with either the LV-scrambled or LV-shRNA37 for 96 h, SAG was added daily at 1 μM for 6 days. A Heat map of reactivity transcripts of all experimental groups analyzed by qPCR, split in PAN reactive, A1-specific and A2-specific reactivity genes. As positive control, HAs were treated with IL-1α, TNFα and C1q for 24 h. Gene expression was normalized to the housekeeping gene GAPDH, quantifying fold change relative to untreated group by the comparative Ct method (n = 4). B Representative immunoblot and quantification of C3 levels in all experimental groups. Equal amounts of protein were immunoblotted and probed with antibodies against C3 and β-actin (as loading control) (n = 5). C The panels on the left show representative images of HAs in the indicated experimental conditions. Cells were stained with specific antibodies against S100β and C3 proteins and with To-Pro-3 to stain the nuclei (blue). Scale bars: 20 μm. The graph on the right shows the fluorescence intensity of C3 + cells in the indicated experimental groups. Each plotted value represents a single cell from at least three independent experiments. D Scratch-wound assay images and quantification of HAs in the different experimental conditions from 0 to 24 h post-scratch (n = 4). The total migration distance was quantified from the acquired images as the distance traveled by HAs from 0 to 24 h. Scale bars: 100 μm. In all graphs, values were normalized to the untreated group (data not plotted). In B data represent mean values ± S.E.M while in C, D data represent values ± S.E.M of the acquired. In A–D data were evaluated using one-way ANOVA followed by Tukey’s post hoc test. In B, C, data from the IL-1α, TNF-α and C1q treated group were compared to the untreated group (data not plotted) using a one-sample t-test