Additional data for "Selenium drives a transcriptional adaptive program to block ferroptosis and treat stroke" by Alim et al.

Paper Title: Selenium drives a transcriptional adaptive program to block ferroptosis and treat stroke These are additional data sets from the related article. 1) Scatter plots for all behavioral data from the paper. GPX4 overexpression scatter plot: (A-B) Forced expression of GPX4 in vivo improves outcome following ICH. Scatterplot of behavioral outcomes in spatial neglect task (n=16; scatter plot) and sensory neglect task (n=16; scatter plot) for up to 14 days post ICH in mice overexpressing GFP or GPX4 in striatum. Selenium treatment scatter plots: (A-B) Scatterplot of behavioral outcomes including spatial neglect task (n=16) and sensory neglect task (n=16) up to 7 days post ICH with or without ICV injection of Se (2.5µM). (C-D) Tat SelPep improves outcomes when given systemically 2h after ICH. Scatterplot of behavioral outcomes in sensory neglect task (n=16) and spatial neglect task (n=16) for up to 14 days post ICH. (E-F) Tat SelPep improves behavioral outcomes with a therapeutic window up to 6 hours. Scatterplot of behavioral outcomes in spatial neglect task ( n=16) and sensory neglect task (n=16) for up to 14 days post ICH in mice given 6 hour post injury IP injections and every day for 7 days of saline, Tat or Tat SelPep (12µg/g). 2) Blots have not been modified with the exceptions of the labels. The file names on the blots correspond to the blots in the figures from the paper. 3) Molecular depletion and validation of exon 5-6 (A-C) and exon 4 (D-F) targeted siRNA of GPX4. Raw blot images are included. 4) (A-D) Differential Interference Contrast (DIC) time-lapse video examples of primary cortical neurons treated with (A) vehicle control, (B) Erastin (5µM), (C) Hemin (80µM), and (D) Hemin+Se (1µM Se). Yellow time bar shows relative hours passed from time of cotreatment, red arrows indicate primary cortical neurons destined to die. Scale bar 100µm. 5) (A-E) ICH modeling in vitro or in vivo shows TUNEL labeling, one feature of ferroptosis. (A-C) HT22 hippocampal neuroblast cells exposed to (A) 100µM Hemin followed by TUNEL labeling, (B)Vehicle control followed by TUNEL labeling, and (C) Hemin without TdT nucleotide treatment followed by TUNEL labeling. White boxes show magnified image and arrows point to TUNEL positive cells. Scale bar 50µM. (D) TUNEL staining (green) of representative brain section from a mouse 7 days after post-saline volume control injection. Inserts represent magnified images with ToPro (blue) and/or TUNEL staining (green). Scale bar 50µM. (E) TUNEL staining (green) of brain section from a mouse 7 days after collagenase injection into the striatum (lCH model). Insert shows ToPro (blue) and/or TUNEL positive cells (green). Scale bar 50µM 6) All hubs from WGCNA data are shown based on RNA-seq conducted in the paper. 7) Caspase inhibitor Z-VAD-FMK or Se protect against ER stress.论文标题：硒介导转录适应性程序以抑制铁死亡并治疗脑卒中 本附属数据集来自相关研究论文，具体如下： 1) 本部分包含论文中所有行为学数据的散点图。 谷胱甘肽过氧化物酶4（GPX4）过表达散点图：(A-B) 体内强制表达GPX4可改善脑出血（ICH）后的神经功能预后。对纹状体过表达绿色荧光蛋白（GFP）或GPX4的小鼠，在脑出血后14天内的空间忽略任务（n=16；散点图）与感觉忽略任务（n=16；散点图）的行为学结果散点图。 硒处理散点图：(A-B) 对脑出血后7天内的小鼠，予以侧脑室注射（ICV）2.5μM硒或不做处理，其空间忽略任务（n=16）与感觉忽略任务（n=16）的行为学结果散点图。(C-D) 脑出血后2小时全身给药Tat硒肽可改善预后，展示其感觉忽略任务（n=16）与空间忽略任务（n=16）在脑出血后14天内的行为学结果散点图。(E-F) Tat硒肽在损伤后6小时给药仍具有治疗窗口，对脑出血后6小时予以腹腔注射（IP）生理盐水、Tat或12μg/g的Tat硒肽，每日给药共7天，其空间忽略任务（n=16）与感觉忽略任务（n=16）在脑出血后14天内的行为学结果散点图。 2) 所有免疫印迹实验结果仅添加标签，未进行其他修饰，印迹文件名称与论文对应图版中的印迹一致。 3) 针对GPX4外显子5-6和外显子4的靶向小干扰RNA（siRNA）的分子敲低及验证实验，原始印迹图像已提供。 4) (A-D) 原代皮层神经元经不同处理后的微分干涉相差（DIC）延时成像示例：(A) 溶剂对照组、(B) 艾拉斯汀（Erastin，5μM）处理组、(C) 氯化血红素（Hemin，80μM）处理组、(D) 氯化血红素+硒（1μM Se）共处理组。黄色时间条标注联合给药后的相对时长，红色箭头指示即将死亡的原代皮层神经元。比例尺为100μm。 5) (A-E) 体外或体内脑出血模型的末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法（TUNEL）染色结果，为铁死亡的特征性标记之一。(A-C) HT22海马神经母细胞分别经(A) 100μM氯化血红素处理后行TUNEL染色、(B) 溶剂对照处理后行TUNEL染色、(C) 氯化血红素处理但未加入TdT核苷酸的阴性对照染色。白色方框标注放大区域，箭头指向TUNEL阳性细胞。比例尺为50μm。(D) 小鼠纹状体注射生理盐水体积对照7天后的脑组织切片TUNEL染色（绿色）结果，插图为使用ToPro（蓝色）和/或TUNEL染色（绿色）的放大图像。比例尺为50μm。(E) 小鼠纹状体注射胶原酶构建脑出血模型7天后的脑组织切片TUNEL染色（绿色）结果，插图展示ToPro（蓝色）和/或TUNEL阳性细胞（绿色）。比例尺为50μm。 6) 基于论文中RNA测序（RNA-seq）数据生成的加权基因共表达网络分析（WGCNA）的所有核心节点（hubs）已展示。 7) 半胱氨酸天冬氨酸蛋白酶抑制剂Z-VAD-FMK或硒可对抗内质网应激。