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）来源星形胶质细胞的核因子κB（nuclear factor kappa-light-chain-enhancer of activated B cells, NF-κB）核转位。(a) 细胞经溶剂对照或五种不同浓度的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均值±标准误（standard error of the mean, SEM）呈现。*P < 0.05，与溶剂对照组存在显著差异；**P < 0.05，与溶剂对照组及1 ng/mL TNF-α组存在显著差异；***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）后进行Tukey多重比较事后检验。放大倍数：100×。校准标尺：200 μm。
图3 人诱导多能干细胞（iPSC）来源星形胶质细胞细胞提取物中的细胞因子表达。细胞经10 ng/mL TNF-α或溶剂对照处理后，分别于1.5、3、4.5、6及24小时评估(a) 白细胞介素6（interleukin-6, IL-6）、(b) 白细胞介素1β（interleukin-1 beta, IL-1β）及(c) 肿瘤坏死因子α（TNF-α）的表达水平。由于未刺激细胞中TNF-α的基础水平低于定量限，(a)、(b)的数据以倍数变化的均值±SEM呈现，(c)的数据以ΔCt值呈现。实验采用4株细胞系，进行三次重复。*P < 0.05；**P < 0.01。N.D.：未检测到。采用单因素方差分析后进行Dunnett事后检验。
图4 人诱导多能干细胞（iPSC）来源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，检测其中细胞因子及BDNF的分泌水平，并与溶剂对照处理组细胞进行比较。(a) 促炎细胞因子：白细胞介素1β（IL-1β）、白细胞介素8（interleukin-8, IL-8）、γ干扰素（interferon gamma, IFN-γ）及肿瘤坏死因子α（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）及白细胞介素6（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）及脑源性神经营养因子（BDNF）。数据以分泌因子浓度（pg/ml）的均值±SEM呈现。实验采用4株细胞系，进行两次重复。*P < 0.05；**P < 0.01；***P < 0.001；ns：无统计学差异。采用独立样本t检验（Unpaired Student´s t-test）。
图5 经5天TNF-α刺激后人诱导多能干细胞（iPSC）来源活化星形胶质细胞的形态学分析。(a) 人iPSC来源星形胶质细胞的免疫荧光染色显微照片，其中波形蛋白（vimentin，红色）、胶质纤维酸性蛋白（GFAP，绿色）及4',6-二脒基-2-苯基吲哚（DAPI，蓝色）被标记；(b) 波形蛋白免疫荧光染色的定量分析；(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免疫荧光染色的定量分析，结果以免疫荧光任意单位（A.U.）表示；(f) DAPI染色的细胞核面积定量分析，以微米平方百分比表示。数据来自4株细胞系的三次重复实验，以均值±SEM呈现。***P < 0.001。采用独立样本t检验。显微照片放大倍数：200×。校准标尺：100 μm。
图6 肿瘤坏死因子α（TNF-α）对人诱导多能干细胞（iPSC）来源星形胶质细胞摄取[³H]D-天冬氨酸的抑制作用。细胞经溶剂对照或10 ng/mL TNF-α处理后，分别于(a) 1天或(b) 5天进行天冬氨酸摄取实验。在加入天冬氨酸前10分钟，添加谷氨酸转运体竞争性抑制剂DL-苏式-β-苄氧基天冬氨酸（DL-threo-beta-benzyloxyaspartate, DL-TBOA）。数据以每分钟计数（counts per minute, cpm）百分比的均值±SEM呈现。(c) 采用乙啶掺入法评估细胞活力：以2%曲拉通裂解细胞作为细胞死亡阳性对照，以在含10%胎牛血清的DMEM/F12培养基中培养的细胞作为细胞活力阳性对照。数据以乙啶掺入百分比（免疫荧光任意单位）的均值±SEM呈现。实验采用3株细胞系，(a)、(b)组进行两次重复，(c)组进行四次重复。*P < 0.05；**P < 0.01；***P < 0.001；ns：无统计学差异。采用单因素方差分析后进行Tukey事后检验。
补充图1 人诱导多能干细胞（iPSC）来源星形胶质细胞中，TNF-α处理不影响持家基因甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase, GAPDH）、输入蛋白8（importin 8, IPO8）及核糖体蛋白P0（ribosomal protein P0, RPLP0）的表达。本实验使用的样本与图3b、3c的数据样本一致。结果以3种持家基因的平均表达值进行标准化。数据来自4株细胞系的三次重复实验，以均值±SEM呈现。ns：无统计学差异。