DOI: 10.6084/m9.figshare.12864419
title: Additional file 2 of Nup133 and ERα mediate the differential effects of hyperoxia-induced damage in male and female OPCs

description: 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 &lt; 0.05, ∗∗p &lt; 0.01, ∗∗∗p &lt; 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 &lt; 0.001, **p &lt; 0.01, *p &lt; 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 &lt; 0.001, **p &lt; 0.01, *p &lt; 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 &lt; 0.05, ∗∗p &lt; 0.01, ∗∗∗p &lt; 0.001, ∗∗∗∗p &lt; 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 &lt; 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 &lt; 0.05, ∗∗p &lt; 0.01, ∗∗∗p &lt; 0.001, ∗∗∗∗p &lt; 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.