DOI: 10.6084/m9.figshare.9249140.v3
title: Quantifications Figures 2-6 & Supplemental Fig. 1 (GraphPad Prism 8.1 files)

description: Figure 2 – TNF-α-induced NF-kB nuclear translocation in human iPSC-derived astrocytes. (a) - Photomicrographs of GFAP (Green), DAPI (Blue) and NF-kB p65 subunit (Red) immunostaining 1 h after exposing cells to vehicle or five different concentrations of TNF-α. Quantification of NF-kB p65 subunit 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 expression in cell extracts from human iPSC-derived astrocytes. (a) – Interleukin-6; (b) – Interleukin-1 beta and (c) – TNF-α expression was assessed after 1.5, 3, 4.5, 6, and 24 hours following cell exposure to 10 ng/mL TNF-α or vehicle. 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; **P &lt; 0.01. N.D. non detected. One-way ANOVA followed by Dunnett post hoc test.<br>Figure 4 – 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 5 – Morphological analysis of human iPSC-derived activated astrocytes following 5 days TNF-α stimulation. (a) - Photomicrographs of human iPSC-derived astrocytes immunostained for vimentin (red), GFAP (green) and DAPI (blue); (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.<br>Supplementary Figure 1 - Expression of housekeeping genes GAPDH, IPO8 and RPLP0, was not affected by TNF-α exposure in human iPSC-derived astrocytes. Samples used in these experiments were also used in data presented on Figures 3b and 3c. Results were normalized by average values of all three housekeeping genes. Graphs represent data from 4 cell lines in experiments performed in triplicates. ns non-significant. Data are presented as means ± SEM.