Figure 1.
Heatmaps of mRNA expression data of MAH infected MDMs after microarray analysis.
Columns represent temporal expression of MDMs at 6, 24 and 48 h p.i. with MAH strains 10091/06 and 104. Colours represent log 2 ratios of the infected cells versus the non-infected control according to the scales shown below. Samples represent a pool of three independent infection experiments. Panel A shows 36 genes possessing 2- to 39-fold (median of all time points) increased expression upon infection, while 10 genes were 2- to 3-fold down-regulated. Panel B and C illustrate mRNAs being temporally induced or repressed after infection. Panel D reflects dysregulated miRNAs after infection, while panel E shows temporally induced miRNAs. An averaged trace of the expression profile is integrated as a white graph by the acuity software (Panel B, C and E). Underlined mRNAs and miRNAs are addressed in the results section.
Table 1.
Differentially expressed protein coding genes after MAH infection.
Table 2.
Distribution of differentially expressed genes among biochemical pathways.
Figure 2.
Regulatory network of MAH infected MDMs deduced from integrated analysis of miRNA-mRNA microarray data.
Negatively correlated miRNA-mRNA interactions were visualised as a network using Cytoscape. This network gives for the first time an theoretical outline of the concerted action of regulating miRNAs (blue triangles) and their potential target mRNAs (green circles) in mycobacterial infection of human macrophages.
Table 3.
Functional enrichment of pathways deduced from integrated miRNA-mRNA analysis.
Figure 3.
Temporal CASP3/CASP7 activity upon MAH infection of MDMs.
MDMs were infected with bacteria and the luminescence signal (relative light units, RLU) proportional to the activity of CASP3/CASP7 was measured. Columns represent the mean of quintuplicate measurements while error bars show the standard deviation. Asterisks indicate statistical significance according to paired t test (***: P<0.001).
Figure 4.
mRNA expression data of MAH infected MDMs after qRT-PCR analysis.
Columns in panel A represent the individual expression of MDMs from three different human donors (Donor 1–3) infected with MAH strains 2514, 10091/06 and 104 as well as E. coli K12 as a control. Colours represent log 2 ratios of the means of triplicate measurements of infected cells versus the non-infected control according to the scales shown below. The box plots in panel B include the data obtained from all three infection experiments considering all three MAH strains. Log 2 ratios below −0.585 and above 0.585 (corresponding to 1.5-fold change) are indicated by the green and red dashed lines and were considered to reflect differential expression.
Figure 5.
miRNA expression data of MAH infected MDMs after miRNA specific qRT-PCR analysis.
The columns show the mean expression of distinct miRNAs from all three donors each measured in triplicates while error bars show the standard deviation. The calculated log 2 ratios of means of all donors relating to each MAH strain and E. coli K12 are shown, respectively. Asterisks indicate statistical significance according to unpaired t test (*: P<0.05; **: P<0.01; ***: P<0.001).
Figure 6.
Regulatory network of MAH infected MDMs deduced from integrated analysis of miRNA-mRNA expression after qRT-PCR experiments.
Negatively correlated miRNA-mRNA interactions were visualised as a network using Cytoscape. Lines represent predicted interactions considering negatively correlating miRNA (blue triangles) and mRNA (green circles) expression data.
Figure 7.
let-7e and miR-29a target CASP3 and CASP7, respectively.
Down-regulation of CASP3 and CASP7 by let-7e and miR-29a was verified using reporter gene assays. Panel A: Identified target sites between miRNAs and both caspases were analysed using RNAhybrid. Panel B: HeLa cells were co-transfected with miRNA mimics and plasmids harbouring the 3′ UTR of CASP3 and 7, respectively. Relative luciferase activity (Luc Gaussia : Luc Cypridina) was determined respective to the a non-sense miRNA mimics serving as a control. The columns show means of normalised luciferase activity each measured in triplicates while error bars show the standard deviation. Asterisks indicate statistical significance between miRNA treated samples and non-sense miRNA treated controls according to paired t test (**: P<0.01).