Quantifications Figures 2-6 & Supplemental Fig. 1 (GraphPad Prism 8.1 files)

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.

图2 – 肿瘤坏死因子α（Tumor Necrosis Factor alpha, TNF-α）诱导人诱导多能干细胞（induced pluripotent stem cell, iPSC）源性星形胶质细胞的核因子κB（Nuclear Factor-kappa B, NF-κB）核转位。(a) 细胞经溶媒或5种不同浓度TNF-α处理1小时后，胶质纤维酸性蛋白（Glial Fibrillary Acidic Protein, GFAP，绿色）、4',6-二脒基-2-苯基吲哚（4',6-Diamidino-2-phenylindole, DAPI，蓝色）及NF-κB p65亚基（红色）的免疫荧光染色显微照片。(b) 细胞核区域与(c) 全细胞区域中NF-κB p65亚基的免疫反应性定量分析，结果以免疫荧光任意单位（arbitrary units, A.U.）表示。(d) NF-κB转位指数（细胞核/全细胞面积比值）。数据以3株细胞系的三次重复实验的平均值±标准误（standard error of the mean, SEM）呈现。*P < 0.05，与溶媒对照组存在显著差异；**P < 0.05，与溶媒对照组及1 ng/mL TNF-α组均存在显著差异；***P < 0.05，与10 ng/mL TNF-α组存在显著差异。采用单因素方差分析（One-way ANOVA）结合Tukey氏事后检验。放大倍数：100×。校准标尺：200 μm。
图3 – 人iPSC源性星形胶质细胞细胞提取物中的细胞因子表达。(a) 白细胞介素6（Interleukin-6, IL-6）；(b) 白细胞介素1β（Interleukin-1 beta, IL-1β）与(c) TNF-α的表达，在细胞经10 ng/mL TNF-α或溶媒处理后的1.5、3、4.5、6及24小时进行检测。由于未刺激细胞中TNF-α的基础水平低于定量限，(a)与(b)的数据以倍数变化的平均值±SEM呈现，(c)的数据以循环阈值差（Cycle threshold difference, ΔCt）呈现。实验采用4株细胞系进行三次重复。*P < 0.05；**P < 0.01。N.D. 未检出（Not Detected）。采用单因素方差分析结合Dunnett氏事后检验。
图4 – 人iPSC源性星形胶质细胞的细胞因子与脑源性神经营养因子（Brain-Derived Neurotrophic Factor, BDNF）分泌情况。细胞经10 ng/mL TNF-α刺激24小时后收集条件培养基，检测其中的细胞因子与BDNF分泌水平，并与溶媒处理组细胞进行对比。(a) 促炎细胞因子：IL-1β、白细胞介素8（Interleukin-8, IL-8）、干扰素γ（Interferon gamma, IFN-γ）及TNF-α；(b) 调节性细胞因子：白细胞介素2（Interleukin-2, IL-2）、白细胞介素4（Interleukin-4, IL-4）及IL-6；(c) 抗炎细胞因子：白细胞介素10（Interleukin-10, IL-10）、白细胞介素13（Interleukin-13, IL-13）及BDNF。数据以分泌因子的浓度（pg/ml）的平均值±SEM呈现。实验采用4株细胞系，条件培养基收集后进行双份重复检测。*P < 0.05；**P < 0.01；***P < 0.001；ns 无统计学意义（non-significant）。采用独立样本学生t检验（Unpaired Student´s t-test）。
图5 – 经5天TNF-α刺激后人iPSC源性活化星形胶质细胞的形态学分析。(a) 人iPSC源性星形胶质细胞的波形蛋白（Vimentin，红色）、GFAP（绿色）及DAPI（蓝色）免疫荧光染色显微照片；(b) 波形蛋白免疫染色的定量分析；(c) 波形蛋白染色细胞的细胞面积定量分析。(d) 星形胶质细胞极化百分比，根据波形蛋白染色标记的长径/短径比值对细胞进行分类。(e) GFAP免疫染色的定量分析，结果以免疫荧光任意单位表示；(f) DAPI染色的细胞核面积定量分析，结果以微米百分比表示。数据以4株细胞系的三次重复实验的平均值±SEM呈现。***P < 0.001。采用独立样本学生t检验。显微照片放大倍数：200×。校准标尺：100 μm。
图6 – TNF-α对人iPSC源性星形胶质细胞摄取[³H]D-天冬氨酸的抑制作用。(a) 细胞经溶媒或10 ng/mL TNF-α处理1天后，或(b) 处理5天后，进行天冬氨酸摄取实验。谷氨酸转运体竞争性抑制剂DL-TBOA在加入天冬氨酸前10分钟添加。数据以每分钟计数（counts per minute, cpm）的百分比的平均值±SEM呈现。(c) 采用乙啶掺入法评估细胞活力。作为细胞死亡阳性对照，用2%曲拉通（Triton）裂解细胞；作为细胞活力阳性对照，检测在含10%胎牛血清的DMEM/F12培养基中培养的细胞。数据以乙啶掺入百分比（免疫荧光任意单位）的平均值±SEM呈现。实验采用3株细胞系，(a)、(b)组进行双份重复检测，(c)组进行四份重复检测。*P < 0.05；**P < 0.01；***P < 0.001；ns 无统计学意义。采用单因素方差分析结合Tukey氏事后检验。
补充图1 – 人iPSC源性星形胶质细胞中，持家基因GAPDH、IPO8及RPLP0的表达不受TNF-α处理的影响。本实验使用的样本与图3b、3c的数据所用样本一致。结果以3种持家基因的平均值进行标准化。图表数据来自4株细胞系的三次重复实验。ns 无统计学意义。数据以平均值±SEM呈现。