Fig 1.
GalCer induces sex-specific alterations in gene expression in Cln3Δex7/8 mouse brain.
Bar chart indicating differences in numbers of significant differentially expressed genes affected by GalCer compared to vehicle treatment in Cln3Δex7/8 male and female mice. A cut-off p-value < 0.05 and fold-change ≥ ±1.3 were assumed to identify genes significantly modulated by GalCer treatment.
Fig 2.
Differentially expressed genes in GalCer versus vehicle-treated male and female Cln3Δex7/8 mouse brain.
2-dimentional heat maps of (a) 66 DEGs in male Cln3Δex7/8 mice, and (b) 30 DEGs in female Cln3Δex7/8 mice. Heat maps show mRNA abundance intensities of differentially expressed genes in profiled samples. Robust Multi-Array Average (RMA) preprocessed data is transformed to z-scores. The legend represents relative over- (red) and under-expression (green). Labeling at the top represents Vehicle control samples and GalCer-treated samples. A cut-off p-value < 0.05 and fold-change ≥ ±1.3 were assumed to identify genes significantly modulated by GalCer treatment.
Fig 3.
Bar chart of GO enrichment scores for DEGs according to biological processes in male/female Cln3Δex7/8 mouse brain.
Gene expression in GalCer- versus vehicle-treated male (a) and female (b) Cln3Δex7/8 mouse brain. Differential expression is assumed with a p-value < 0.05, and a gene count > 2 of the DEGs. Functional categories of DEGs were obtained using GO annotations from the Partek Genomics Suite classification system.
Fig 4.
Forest plot of GO for DEGs in biological processes in male Cln3Δex7/8 mouse brain.
Gene expression in GalCer- versus vehicle-treated Cln3Δex7/8 male mouse brain. The criterion for differential expression assumes a p-value < 0.05. Forest plots of biological processes including (a) developmental, (b) cellular, and (c) behavioral processes depict percentage of down- (green) or up-regulated (red) genes in each category.
Fig 5.
Forest plot of GO for DEGs in biological processes in female Cln3Δex7/8 mouse brain.
Gene expression in GalCer versus vehicle-treated female Cln3Δex7/8 mouse brain. The criterion for differential expression assumes a p-value < 0.05. Forest plots of biological processes of (a) developmental, (b) cellular, and (c) behavioral processes depict percentage of down- (green) or up-regulated (red) genes in each category.
Fig 6.
Gene-disease relationship network of significant DEGs in GalCer- versus vehicle-treated male Cln3Δex7/8 mice.
Pathway Studio software generated network interactions between diseases and most significant DEGs in male Cln3Δex7/8 mouse brain (p-value < 0.05, stringency ≥ ± 1.3 fold-change in expression). Depiction of genes is by their gene symbols. Upregulated genes are highlighted with a red halo and downregulated genes are highlighted with a green halo. Indirect regulation is indicated by dotted gray lines.
Fig 7.
Gene-disease relationship network of significant DEGs in GalCer- versus vehicle-treated female Cln3Δex7/8 mice.
Pathway Studio software generated network interactions between diseases and most significant DEGs in female Cln3Δex7/8 mouse brain (p-value < 0.05, stringency ≥ ± 1.3 fold-change in expression). Depiction of genes is by gene symbols. Upregulated genes are highlighted with a red halo and downregulated genes are highlighted with a green halo. Indirect regulation is indicated by dotted gray lines.
Fig 8.
Validation of microarray analysis results with qRT-PCR in male and female Cln3Δex7/8 mouse brain.
Relative mRNA expression measured by qRT-PCR. (a) The following differentially expressed genes in male Cln3Δex7/8 mouse brains were randomly chosen: Atp6v0a1, Braf, Dnm3, Prkacb, Gabbr, Grm1, and C1qb. Values are means of the fold changes normalized to Gapdh mRNA expression, with standard errors represented by vertical bars.*p-value < 0.05 and ** p-value < 0.01 by Student’s t-test (n = 4). (b) The following differentially expressed genes in female Cln3Δex7/8 mouse brains were randomly chosen: Fos, Prlr, and Fdft1. Values are means of the fold changes normalized to Gapdh mRNA expression, with standard errors represented by vertical bars. *** p-value < 0.001 and **** p-value < 0.0001 by Student’s t-test (n = 4).
Fig 9.
Comparative analysis of DEGs in male and female Cln3Δex7/8 mouse brain.
Depiction of DEGs selected in male and female Cln3Δex7/8 mouse brain based on gene expression in GalCer compared to vehicle-treated Cln3Δex7/8 mice of both genders. The criterion for differential expression was a p-value < 0.05, and a stringency ≥ ± 1.3-fold change in expression. (a) A total number of 96 genes, with 65 and 29 genes exclusive for male and female Cln3Δex7/8 mouse brain, respectively, shown. One gene appears in the overlapping region, common to both datasets. (b) Fold change and p-value of the gene (Ddit4) common to male and female datasets. (c) Schematic representation of the effect of GalCer on the function of Ddit4 in brain.