Gene expression profiling of dexamethasone effects in multiple tissues

Description

Methods (English)

C57BL/6J mice were killed by decapitation 4 h after a single dexamethasone (DEX, 10 mg/kg) i.p. injection, while saline-treated mice (10 ml/kg) served as the control group. The dose of DEX was based on our and others previous experiments, paying particular attention to avoiding systemic toxic effects and overcoming the blockade of the blood-brain barrier. Tissue samples (adrenal cortex, perigonadal adipose tissue, hypothalamus, liver, kidney, anterior thigh muscle, pituitary gland, spleen, and lungs) were fixed. RNA was isolated using the RNeasy Mini Kit and further purified following the manufacturer's protocol. RNA from two mice was randomly pooled to prepare samples for each microarray. Each cRNA sample was hybridized overnight to the MouseWG-6 v2 BeadChip arrays (Illumina) in a multiple-step procedure according to the manufacturer's instructions. Raw microarray data was generated using BeadStudio v3.0 (Illumina). The obtained signal was taken as the measure of mRNA abundance derived from the gene expression level. Statistical analysis of the results was performed using two-way ANOVA (for tissue and treatment) followed by Tukey's HSD post-hoc tests (where appropriate). The false discovery rate (FDR) was estimated using the Benjamini and Hochberg method. We employed a comprehensive statistical approach to evaluate the differential impact of DEX across various tissues. Overrepresentation analyses were performed using Enrichr, a comprehensive gene set enrichment analysis tool that integrates data from multiple genomic resources. Three datasets containing lists of (I) GR-dependent genes, (II) genes linked to metabolic traits, and (III) genes associated with various human phenotypes were prepared based on external resources.

Table of contents (English)

Supplementary files:

1. Supplementary Table 1: A table listing the comprehensive set of 585 DEX-regulated transcripts and the lists of genes from patterns A - P. Each of these is available as a separate spreadsheet. The results of two-way ANOVA (for drug and time) are presented. The false discovery rate (padj) was estimated using the Benjamini and Hochberg method. (XLS).

2. Supplementary Table 2: A data file providing lists of studies investigating GCs effects on gene transcription in various tissues and cell types. The second spreadsheet contains a table listing the complete chi-square test results of overlap (padj < 0.01, and at least three overlapping genes) between the identified GR-dependent gene patterns and gene lists from the literature. The top results of gene overlap are presented in Figure 4. (XLS).

3. Supplementary Table 3: A table listing the complete results of the in silico analysis of promoter regions of DEX-regulated genes. The analyses were performed on lists of genes that correspond to patterns A-P, as well as lists of genes with increased (UP) and decreased (DOWN) mRNA abundance levels in response to DEX. The genes are listed in Supplementary Table 1. Gene lists were used as input to Enrichr to identify overrepresented transcription factor binding sites (track ChEA 2022). Only results with nominal p-values below 0.05 and involving at least two genes were included in the file. (XLS).

4. Supplementary Table 4: A table listing the complete results of the GO analysis presented in the manuscript (Functional classification). The analyses were performed on lists of genes that correspond to patterns A-P, as well as lists of genes with increased (UP) and decreased (DOWN) mRNA abundance levels in response to DEX. The genes are listed in Supplementary Table 1. Gene lists were used as input to Enrichr to identify overrepresented biological processes (track GO Biological Process 2023). Only results with nominal p-values below 0.05 and involving at least two genes were included in the file. (XLS).

5. Supplementary Table 5: A table listing the overlap with drug gene-expression signatures from previously published studies. The analyses were performed on lists of genes that correspond to patterns A-P, as well as lists of genes with increased (UP) and decreased (DOWN) mRNA abundance levels in response to DEX. The genes are listed in Supplementary Table 1. Gene lists were used as input to Enrichr to identify overrepresented biological processes (track DSigDB). Only results with nominal p-values below 0.05 and involving at least two genes were included in the file. (XLS).

6. Supplementary Table 6: A data file providing tables of the complete chi-square test results of overlap (padj < 0.01, and at least three overlapping genes) between the identified GR-regulated gene clusters and human phenotypic traits (Figure 5A, Spreadsheet 1) or metabolism-related traits (Figure 5B, Spreadsheet 2). The lists include symbols of overlapping genes. The top results of gene overlap are presented in Figure 5. (XLS).