FIGURE 5

FIGURE 5

Oxidative stress is present in malformed cells in TSC cortical tubers and stimulates SPI1 expression in vitro. Expression of iNOS could be detected exclusively in malformed cells in FCD 2b and TSC tissue. Co‐localization of SPI/PU.1 and iNOS was found in some malformed cells (arrows in A, B and A₂). Moreover, iNOS expression was often found in cells that were in close proximity to SPI1/PU.1‐expressing cells (A₁, B₁) that wrapped around each other (A, B). Analysis of the OS markers GPx1, SOD1, and TXNRD1 showed higher expression in TSC tissue compared to control (C). Human fetal astrocytes did not display higher SPI1 expression when exposed to IL‐1β, but H₂O₂ in a time‐dependent manner (D, E). SH‐SY5Y neuroblastoma cells exposed to H₂O₂ displayed SPI1 induction, similar to astrocytes (F). In vitro, TSC‐derived astrocytes displayed higher expression of SPI1 when exposed to H₂O₂, which was independent of mTOR inhibition via rapamycin (G). Scale bars: 50 µm in A_, 25 µm in A₁, 20 µm in B₁, 10 µm in A₂, arrowheads = iNOS single positive cells, arrows = co‐localized cells. (C and D) Mann–Whitney U test. (E–G) Kruskal–Wallis test with Dunn's post hoc. (C) Data are expressed relative to expression in autopsy control and displayed as Tukey's box plot. (D–G) Data are expressed as bar graphs with SD; *p < 0.05, **p < 0.01, ***p < 0.001. (C) n = 8 autopsy control versus n = 10 TSC samples. (D–G) n = 3 independent cultures in duplicates