Figure 1.
Principal component analysis of microarray data.
PCA was made over normalized expression levels of expressed genes in the array (detection P-Value <0.01 in at least one sample). The first 3 principal components accounted for the 85% of explained variance and clustered apart samples coming from the different genotypes and treatments. Black: IDA. Red: IDA+LPS. Green: Tmprss6 KO. Blue: Tmprss6 KO+LPS.
Figure 2.
Heat map of clustered biological terms highlighted by differentially expressed genes in the “Genotype contrasts”.
The heat map represents semantic similarity among gene ontology (GO) Biological Process (BP) terms. Rows and columns show the list of enriched GO BP terms derived from term enrichment analysis of Genotype significant genes. The colors represent the semantic distances calculated using GOSemSim Bioconductor package. Yellow-red clusters identify groups of terms sharing semantic similarity about biological processes.
Figure 3.
Representations of Kegg pathways enriched in the “Genotype contrast”.
A) KEGG pathways derived from term enrichment analysis. Bars represent −10*log10(P-Value). The dotted line shows significance cut-off at enrichment analysis, which corresponds to a P-Value of 0.05. B) Representation of the KEGG Toll-Like Receptor Signaling Pathway, showing up-regulated (red boxes) and down-regulated (green boxes) genes.
Figure 4.
Heat map of selected genes (basal condition).
The heat map represents the hierarchical clustering of 49 genes being differentially expressed according to the “Interaction contrast” (adjusted P-Value <0.05 and |log2ratio| >1). The expression level of each gene has been standardized by subtracting the gene’s mean expression and then dividing by the standard deviation across all samples. This scaled expression value, denoted as the Row Z-score, is plotted in red-blue scale color, with red indicating high expression.
Table 1.
Genes differentially regulated under basal conditions in the liver of Tmprss6−/− vs IDA mice.
Figure 5.
Analysis of liver BMP-SMAD, STAT3 and NF-kB proteins activation.
Livers were dissociated as described in the “Material and Methods” section; extracts were subjected to SDS-PAGE and Western Blot performed using anti-Phosphorylated-SMAD1/5/8 (P-SMAD), anti-Phosphorylated-STAT3 (P-STAT3), anti-NF-kB p100, and anti-NF-kB p65. Protein levels were quantified by densitometric analysis of P-SMAD, P-STAT3, NF-kB p100 and NF-kB p65 specific bands, normalized to actin. 1 and 2 refers to liver extracts from two different mice. The numbers under the panels indicate arbitrary densitometric unit.
Figure 6.
Genes differentially expressed under basal conditions.
Total liver RNA was isolated from Tmprss6 KO and IDA mice. mRNA expression was quantified by TaqMan qRT-PCR. Hprt1 was used as the housekeeping gene. mRNA expression ratio was normalized to an IDA mean value of 1. Error bars indicate Standard Error.; ns, not significant; *P<0.05; **P<0.01; and ***P<0.001. White bar: IDA mice; grey bar: Tmprss6 KO mice.
Figure 7.
Genes differentially expressed after LPS treatment.
Total liver RNA was isolated from Tmprss6 KO and IDA mice treated with LPS to induce acute inflammation (3 mice each group). TaqMan qRT-PCR was used to quantify mRNA expression and Hprt1 was used as the housekeeping gene. mRNA expression ratio was normalized to an IDA mean value of 1. Error bars indicate Standard Error.; ns, not significant; *P<0.05; **P<0.01; and ***P<0.001. White bar: IDA mice; grey bar: Tmprss6 KO mice.
Table 2.
Genes differentially regulated by LPS in the liver of Tmprss6−/− vs IDA mice.
Figure 8.
Heat map of clustered biological terms highlighted by differentially expressed genes in the “Interaction contrasts”.
The heat map represents semantic similarity among gene ontology (GO) Biological Process (BP) terms. Rows and columns show the list of enriched GO BP terms derived from term enrichment analysis of Interaction significant genes. The colors represent the semantic distances calculated using GOSemSim Bioconductor package. Yellow-red clusters identify groups of terms sharing semantic similarity about biological processes.
Figure 9.
Heat map of selected genes (“Interaction contrast”).
The heat map represents the hierarchical clustering of 59 genes being differentially expressed according to the “Interaction contrast” (adjusted P-Value <0.05 and |log2ratio| >1). The expression level of each gene has been standardized by subtracting the gene’s mean expression and then dividing by the standard deviation across all samples. This scaled expression value, denoted as the Row Z-score, is plotted in red-blue scale color, with red indicating high expression.
Figure 10.
Modulation of representative genes by iron/hepcidin.
7 weeks old mice (n = 4 per group) were maintained an iron deficient (IDA, white bar), iron balanced (IB, light grey bar) and iron loaded (IL, dark grey bar) diet for 3 wks. Liver mRNA expression was measured by TaqMan qRT-PCR. Hprt1 was used as the housekeeping gene. mRNA expression ratio was normalized to an IB mean value of 1. Error bars indicate Standard Error; ns, not significant; *P<0.05 and **P<0.01.