Relative quantification of the expression level of the protease genes.

<p>Data from three independent total mRNA extractions of <i>E. coli</i> AG100 physiologically adapted to increasing concentrations of TET compared to its parental non-induced strain grown in absence of TET as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000365#s3" target="_blank">Materials and Methods</a>. A ratio of 1 corresponds to no alterations in expression compared with untreated control cells. Values were corrected for standard deviation range.</p

The effect of serial passages in a constant concentration of isoniazid (0.1 µg/ml) on the INH MIC and the number of days required for detection of growth.

<p><b>Legend:</b> INH: isoniazid; RIF: rifampicin; INH (a)/(b): adaptation processes to isoniazid A and B, respectively.</p

Relative quantification of phagosomal acidification in <i>M. bovis</i> BCG-GFP-infected macrophages stained with LysoTracker Red and analysed by flow cytometry.

<p> Human macrophages were infected with <i>M</i>. <i>bovis</i> BCG-GFP, treated with verapamil (VP), thioridazine (TZ), chlorpromazine (CPZ), flupenthixol (FPX), and haloperidol (HAL). Data was analysed by flow cytometry using an easyCyte^™ 5HT flow cytometer. A) Bars graph: quantification of the increase on the overall number of LysoTracker Red stained vesicles per cell (average fluorescence intensity); B) cytograms for (i), unstained and non-treated infected macrophages; (ii), stained and non-treated infected macrophages; and (iii) to (vii), stained and infected macrophages treated with VP, TZ, CPZ, FPX and HAL. VP was tested at 10 μg/ml; TZ at 2.5 μg/ml; CPZ, HAL and FPX at 1.25 μg/ml. Significance of the results was tested using Student’s t-test and were considered highly significant, ***<i>P</i> <0.001.</p

Relative expression of outer membrane proteins, regulators and inner membrane transporter genes.

<p>Data from three independent total mRNA extractions of <i>E. coli</i> AG100 physiologically adapted to increasing concentrations of TET compared to its parental non-induced strain grown in the absence of TET as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000365#s3" target="_blank">Materials and Methods</a>. A ratio of 1 corresponds to no alterations in expression compared with untreated control cells. Values were corrected for standard deviation range.</p

Average quantification of the relative expression level,by RT-qPCR,of the genes that code for efflux pumps in <i>M. tuberculosis</i>exposed to isoniazid.

<p>Average quantification of the relative expression level,by RT-qPCR,of the genes that code for efflux pumps in <i>M. tuberculosis</i>exposed to isoniazid.</p

Determination of cathepsin B activity.

<p> Cathepsin B activity was evaluated in human monocyte-derived macrophages infected and non-infected with <i>M</i>. <i>tuberculosis</i> H37Rv and non-treated and treated with verapamil (VP), thioridazine (TZ), chlorpromazine (CPZ), flupenthixol (FPX) and haloperidol (HAL). VP was tested at 10 μg/ml; TZ at 2.5 μg/ml; CPZ at 1.25 μg/ml; HAL at 1.25 μg/ml; FPX at 1.25 μg/ml. The results were considered highly significant, **<i>P</i> < 0.01.</p

Primers used in this study.

<p>Primers used in this study.</p

Immunodetection of the outer membrane proteins.

<p>The detections were carried out using antisera prepared against OmpC porin (A), OmpF porin (B), antigenic peptide located inside the internal loop 3 porin (C), OmpA (D) and OmpX (E) respectively. Immunodetection were carried out with total cell extracts from non-induced AG100 (1, 2) and 10 mg/mL TET-induced (3, 4) strains grown in LB and MH media (odd and even lanes respectively).</p

Average quantification of the relative expression level,by RT-qPCR,of the genes that code for efflux pumps in <i>M. tuberculosis</i>exposed to rifampicin.

<p>Average quantification of the relative expression level,by RT-qPCR,of the genes that code for efflux pumps in <i>M. tuberculosis</i>exposed to rifampicin.</p

Tetracycline activation cascade of <i>E. coli</i> resistance physiological adaptation.

<p>Broken arrows indicate the activation in 1 and 2 over-expression of specific gene (direct TET pressure effect), in 3, the regulation by induced regulators (second level of control), in 4 the effect of activated genes coding for membrane proteins (third level of effect). Thick arrows (5) illustrate the effect of over-production of OMPs and proteases.</p