Principal component analysis for all individual control and <i>M. bovis</i>-challenged MDM at the 2 hour, 6 hour and 24 hour time points.

<p>Principal component (PC) 1 and PC2 are shown (accounting for 23.31% and 17.05% of the total variation, respectively). PCA was performed using data for all genes whose probes passed the data filtering process with Euclidean distance as the distance metric.</p

The number of significantly differentially expressed genes at each time point post-<i>M. bovis</i> challenge.

<p>The number of upregulated and downregulated genes in the <i>M. bovis</i>-challenged MDM relative to the control MDM at each time point are shown (adjusted <i>P</i>-value 0.05).</p

Differential gene expression in the TLR signalling pathway 2 hours post-<i>M. bovis</i> challenge.

<p>Genes within the TLR signalling pathway showing differential expression are highlighted in colour. Colour intensity indicates the degree of upregulation (red) or downregulation relative to the control MDM. Grey shading indicates genes that were not differentially expressed; white shading represents genes in the pathway not represented on the microarray; mycobacterial PAMPs are shaded orange.</p

Schematic depicting the experimental design used in the current study.

<p>MDM were cultured in 24-well tissue culture plates (2×10⁵ cells per well) and challenged with <i>M. bovis</i> (MOI 2∶1). RNA was extracted from <i>M.bovis</i>-challenged and non-challenged control MDM at three time points post-challenge: 2 hours, 6 hours and 24 hours. In addition, RNA was extracted from a 0 hour non-challenged control to assess potential non-experimental changes in gene expression. The MDM lysates from replicate tissue culture wells (shaded) were pooled for RNA extraction. Global gene expression for the control and <i>M. bovis</i>-challenged MDM was analysed using the Affymetrix® GeneChip® Bovine Genome Array.</p

Differential gene expression associated with apoptosis 6 hours post-<i>M. bovis</i> challenge.

<p>Genes associated with apoptosis showing differential expression are highlighted in colour. Colour intensity indicates the degree of upregulation (red) or downregulation relative to the control MDM. Grey shading indicates genes that were not differentially expressed; white shading represents genes in the pathway not represented on the microarray.</p

Real time qRT-PCR analysis of the <i>IL1B</i>, <i>IL6</i>, <i>NFKB2</i> and <i>TNF</i> genes following <i>M. bovis</i> challenge.

<p>Log₂ fold-changes in expression in the <i>M. bovis</i>-challenged MDM relative to the non-challenged control MDM at all three time points are shown. For comparison, the expression profiles for these genes based on the microarray data are also shown. The significance of the fold-changes in expression for each gene based on the real time qRT-PCR analysis only are denoted by asterisks in the figure (*<i>P</i>≤0.05, **<i>P</i>≤0.01, ***<i>P</i>≤0.001). The fold-changes calculated for each gene from the microarray data in the <i>M. bovis</i>-challenged MDM for each gene at each time point were significant (adjusted <i>P</i>-value ≤ 0.05). In addition, the log₂ fold-change in expression for the control MDM at each time point relative to the 0 hour control MDM are also shown for both the microarray and real time qRT-PCR data; no significant differences in gene expression between the control MDM relative to the 0 hour control was observed for these genes at each time point (<i>P</i>≥0.05).</p

Differential gene expression associated with intracellular pathogen recognition receptors 24 hours post-<i>M. bovis</i> challenge.

<p>Genes associated with intracellular PRR signalling showing differential expression are highlighted in colour. Colour intensity indicates the degree of upregulation (red) or downregulation relative to the control MDM. Grey shading indicates genes that were not differentially expressed; white shading represents genes in the pathway not represented on the microarray; viral PAMPs are shaded orange.</p