Additional file 2 of Nup133 and ERα mediate the differential effects of hyperoxia-induced damage in male and female OPCs

Additional file 2: Figure S1. Proteins involved in cell adhesion and migration downregulated in male OPCs. (A) Immunoblot analysis of RhoA protein showing downregulation in male OPCs post 24 h 80%O2 treatment. (B) Heat-map representation of cell adhesion related proteins that were dysregulated in male and female derived OPCs post 24 h 80%O2 treatment in comparison to 3%O2 (normoxia) controls. Mapped expression ratios are depicted with a color scale as shown in the figure, such that highly downregulated proteins are indicated in red, intermediate in yellow, and highly upregulated proteins in green. Proteins are sorted according to Gene ontology (biological process). Dark outlined cells represent the significant proteins in each group. The cut off p value being 0.07. Data are representative of five independent experiments. (C) Independent intensities of Rac1, Mapk1, Map 2k1 and Arpc1b plotted from. MS results showing a significant downregulation in male derived-OPCs post hyperoxia. Data are representative of three experiments. Bars and error represent mean ± SEM of replicate measurements. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t test). Figure S2. Changes in nuclear envelope proteins in OPCs post hyperoxia. (A) Western blot analysis of male and female OPCs with anti-Nup-50 and anti-Lamin B1 antibodies under normal (3%O2) conditions and post 24 h 80%O2 treatment, showing a significant decrease in expression in the male OPCs. Whereas in female OPCs, Nup50 showed a significant upregulation post hyperoxia. ***p < 0.001, **p < 0.01, *p < 0.05 (Student’s t test), n = 3. Values are means ± SEM. (B) mRNA expression of Nup210 showing downregulation in male OPCs and upregulation in female OPCs post hyperoxia. ***p < 0.001, **p < 0.01, *p < 0.05 (Student’s t test), n = 3. Values are means ± SEM. (C) Intensities of Lamin B1, Lamin B2, Pre-Lamin A/C, Nup210, Nup155 and Nup98 plotted from the mass spectrometry results show a significant downregulation of Lamin B1, Lamin B2, Nup155 and Prelamin A/C in male derived OPCs post hyperoxia. For Nup210 and Nup98 a similar trend in both cell groups was observed. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (Student’s t test), n = 3. Values are means ± SEM. Figure S3. Inherent protein profile differences between male and female OPCs under normal conditions. (A) Functional categorization of significantly different proteins in male and female OPCs under normal conditions using IPA. Bar graphs depict the most extensively enriched biological processes among the significantly different proteins. Cut off p value being 0.05. (B) STRING Protein interaction analysis of the proteins detected to be significantly different (p < 0.05) in the male vs female (normoxia) group from the MS data, with an interaction score of high confidence (0.700), showing proteins related to energy and metabolism, protein synthesis and a few cytoskeletal proteins to be differentially expressed in male and female cells. Figure S4. Nup133 ChIP-Seq additional results and validation. (A) Pie chart showing proportions of genomic landmarks corresponding to Nup133-bound targets. (B) De novo motif analysis of Nup133-binding regions identified different motifs with the highest significance ranking in male and female groups. The q-values for all the presented motifs were determined as 0.01. ChIP-Seq data are representative of four independent experiments. (C) mRNA expression validation of Cnp and Egr2 showing downregulation in male OPCs post hyperoxia. Whereas Hes 5 that acts as a transcription repressor and a negative regulator of oligodendrocyte differentiation is upregulated in male OPCs post hyperoxia. Data are representative of three independent experiments. Bars and error represent mean ± SEM of replicate measurements. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (Student’s t test). Figure S5. Validation of Nup133 antibody and ChIP-Seq targets by ChIP-qPCR. (A) Nup133 immunoprecipitation (IP) detected by immunoblot in male and female derived OPCs without any treatment. Positive control shown here is cell lysate obtained from male OPCs and negative control is the pull down product obtained from IP performed using control IgG antibody. (B) Western blot detection of Nup133 bands in cell lysates prepared from untreated OPCs. (C) Representative immunofluorescence images of OPCs stained for Nup133 at different focal plains. Scale bar represents 25 μm. (D) IGV genome browser tracks showing enrichment peaks for each respective target. Primers were designed specifically ON-target covering the highest peak areas. Graphs depicting enrichment over total genomic input (%Input) for male, female and mock IgG (IgG) control conditions for 6 chosen gene targets identified by ChIP-Seq. Results for all ChIP-qPCR data were generated from three independent experiments.

附加文件2：补充图S1。男性少突胶质前体细胞（OPCs）中参与细胞黏附与迁移的下调蛋白。(A) 蛋白质免疫印迹（Western blot）分析显示，经80%氧气处理24小时后，男性OPCs中的RhoA蛋白表达下调。(B) 热图展示了相较于3%氧气（常氧）对照组，经80%氧气处理24小时后，雌雄来源OPCs中失调的细胞黏附相关蛋白。映射的表达比值以图示色阶呈现：高度下调蛋白以红色标示，中度变化以黄色标示，高度上调蛋白以绿色标示。蛋白按基因本体论（生物过程）排序。带深色边框的单元格代表各组中的显著差异蛋白，显著性 cutoff 值为0.07。实验数据来自5次独立重复实验。(C) 基于质谱（mass spectrometry, MS）结果绘制的Rac1、Mapk1、Map2k1及Arpc1b的独立强度曲线，显示高氧处理后男性来源OPCs中这些蛋白显著下调。实验数据来自3次独立重复实验。柱状图与误差棒代表重复测量的均值±标准误（standard error of the mean, SEM）。*p < 0.05，**p < 0.01，***p < 0.001（学生t检验）。

补充图S2：高氧处理后OPCs中核被膜蛋白的变化。(A) 分别在正常（3%O₂）条件及80%氧气处理24小时后，使用抗Nup-50与抗Lamin B1抗体对雌雄OPCs进行蛋白质免疫印迹分析，结果显示男性OPCs中这两种蛋白的表达显著降低；而女性OPCs中Nup50的表达在高氧处理后显著上调。***p < 0.001，**p < 0.01，*p < 0.05（学生t检验），n=3。数值以均值±标准误表示。(B) Nup210的mRNA表达量显示，高氧处理后男性OPCs中其表达下调，女性OPCs中则上调。***p < 0.001，**p < 0.01，*p < 0.05（学生t检验），n=3。数值以均值±标准误表示。(C) 基于质谱结果绘制的Lamin B1、Lamin B2、Pre-Lamin A/C、Nup210、Nup155及Nup98的强度变化，显示高氧处理后男性来源OPCs中Lamin B1、Lamin B2、Nup155及Pre-Lamin A/C显著下调；Nup210与Nup98在两类细胞中呈现相似的变化趋势。*p < 0.05，**p < 0.01，***p < 0.001，****p < 0.0001（学生t检验），n=3。数值以均值±标准误表示。

补充图S3：正常条件下雌雄OPCs的固有蛋白谱差异。(A) 使用Ingenuity通路分析（IPA）对正常条件下雌雄OPCs中显著差异表达的蛋白进行功能分类，柱状图展示了这些差异蛋白中最显著富集的生物过程。显著性cutoff值为0.05。(B) 基于质谱数据，对常氧条件下雌雄OPCs组中显著差异表达（p < 0.05）的蛋白进行STRING蛋白互作分析，互作评分设置为高置信度（0.700），结果显示与能量代谢、蛋白质合成及部分细胞骨架蛋白相关的蛋白在雌雄细胞中存在差异表达。

补充图S4：Nup133染色质免疫沉淀测序（ChIP-Seq）的补充结果与验证。(A) 饼图展示了与Nup133结合的靶标对应的基因组区域标注比例。(B) 对Nup133结合区域进行从头基序分析，鉴定出雌雄两组中显著性排名最高的不同基序，所有展示基序的q值均为0.01。ChIP-Seq数据来自4次独立重复实验。(C) 对Cnp与Egr2的mRNA表达进行验证，结果显示高氧处理后男性OPCs中二者表达下调；而作为转录抑制因子及少突胶质细胞分化负调控因子的Hes5，在男性OPCs中高氧处理后表达上调。实验数据来自3次独立重复实验。柱状图与误差棒代表重复测量的均值±标准误。*p < 0.05，**p < 0.01，***p < 0.001，****p < 0.0001（学生t检验）。

补充图S5：通过染色质免疫沉淀实时定量PCR（ChIP-qPCR）验证Nup133抗体及ChIP-Seq靶标。(A) 未经过任何处理的雌雄来源OPCs中，通过免疫印迹检测到的Nup133免疫沉淀（IP）产物。阳性对照为男性OPCs的细胞裂解液，阴性对照为使用对照IgG抗体进行免疫沉淀的下拉产物。(B) 对未处理OPCs制备的细胞裂解液进行蛋白质免疫印迹，检测Nup133条带。(C) 不同聚焦平面下OPCs的Nup133免疫荧光染色代表性图像，比例尺为25 μm。(D) IGV基因组浏览器轨迹图展示了各对应靶标的富集峰。引物设计特异性靶向覆盖最高峰值区域。绘制了6个经ChIP-Seq鉴定的靶基因在雌雄样本及模拟IgG（IgG）对照条件下，相对于总基因组输入量的富集情况（%Input）。所有ChIP-qPCR数据均来自3次独立重复实验。