Quantifications Figure 2-6 (GraphPad Prism 8.1 files)

Figure 2 – TNF-α-induced NF-kB nuclear translocation in human iPSC-derived astrocytes. Quantification of NF-kB immunoreactivity in both (b) – Nuclei and (c) – Whole cell area, which were expressed in arbitrary units of immunofluorescence (A.U.). (d) – The NF-kB translocation index (nuclei/whole cell area ratio). Data are presented as means ± SEM of experiments performed in triplicates from 3 cell lines. *P &lt; 0.05, different from vehicle; **P &lt; 0.05, different from vehicle and 1 ng/mL TNF-α. ***P &lt; 0.05, different from 10 ng/mL TNF-α. One-way ANOVA followed Tukey´s post hoc test. Magnification: 100 x. Calibration bar: 200 μm.<br>Figure 3 – Cytokines and BDNF secretion from human iPSC-derived astrocytes. Conditioned media were collected after stimulating cells during 24 h with 10 ng/mL TNF-α. Cytokines and BDNF secretion was measured in the conditioned media and compared with cells treated with vehicle. (a) - Pro-inflammatory cytokines: Interleukin-1 beta (IL-1β), Interleukin-8 (IL-8), Interferon gamma (IFN-γ) and Tumor necrosis factor alpha (TNF-α); (b) - modulatory cytokines: Interleukin-2 (IL-2), Interleukin-4 (IL-4) and Interleukin-6 (IL-6); (c) - anti-inflammatory cytokines Interleukin-10 (IL-10), Interleukin-13 (IL-13) and Brain-derived neurotrophic factor (BDNF). Data are presented as means ± SEM of concentrations in (pg/ml) of secreted factors. Conditioned media were collected from 4 cell lines and the experiments were performed in duplicates. *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; ns - non-significant. Unpaired Student´s t-test.<br>Figure 4 – Cytokines expression in cell extracts from human iPSC-derived astrocytes. (a) – Interleukin-6; (b) – Interleukin-1 beta and (c) – TNF-α expression was assessed 24 h after exposing cells to vehicle or 10 ng/mL TNF-α. Data are presented as means ± SEM of fold change for (a) and (b) and Ct (c) since the basal levels of TNF-α were below the limit of quantification in non-stimulated cells. Experiments were performed in triplicates from 4 cell lines. *P &lt; 0.05; N.D. non detected. Unpaired Student´s t-test.<br>Figure 5 – Morphological analysis of human iPSC-derived activated astrocytes following 5 days TNF-α stimulation. (b) – Quantification of vimentin immunostaining; (c) – Quantification of cell area in vimentin-stained cells. (d) – Percentage of astrocyte polarization, which cells were classified according to increase of length/width ratio of vimentin-stained labeling. (e) - Quantification of GFAP immunostaining expressed in arbitrary units of immunofluorescence (A.U); (f) - Quantification of DAPI-stained nuclei areas expressed as percentage of micrometers. Data are presented as means ± SEM from 4 cell lines in experiments performed in triplicates. ***P &lt; 0.001. Unpaired Student´s t-test. Photomicrographs magnification: 200x. Calibration bar: 100 μm.<br>Figure 6 – Impairment of [3H] D-aspartate uptake by TNF-α in human iPSC-derived astrocytes. Aspartate uptake was carried out (a) – 1 day or (b) – 5 days after exposing cells to vehicle or 10 ng/mL TNF-α. The competitive inhibitor of glutamate transporters DL-TBOA was added 10 minutes prior to aspartate. Data are presented as means ± SEM of the percentage of counts per minute (cpm). (c) – Cell viability was evaluated by ethidium incorporation. As a positive control for cell death, cells were lysed with Triton 2 %. As a positive control for cell viability, cells grown in DMEM/F12 with 10 % SFB were also evaluated. Data are presented as means ± SEM of the percentage of ethidium incorporation (arbitrary units of fluorescence). Data from 3 cell lines and experiments were performed in duplicates (a), (b) and quadruplicates (c). *P &lt; 0.05; **P &lt; 0.01; ***P &lt; 0.001; One-way ANOVA followed by Tukey post hoc test. ns – Non-significant.