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

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

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

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

Molecular characterization,resistance pattern and MIC values of the antibiotics and inhibitors for the nine <i>M. tuberculosis</i>strains.

<p>Molecular characterization,resistance pattern and MIC values of the antibiotics and inhibitors for the nine <i>M. tuberculosis</i>strains.</p

Synergistic activity between the ion channel blockers and isoniazid,rifampicin,amikacin or ofloxacin,against the nine <i>M. tuberculosis</i>strains.

<p>Synergistic activity between the ion channel blockers and isoniazid,rifampicin,amikacin or ofloxacin,against the nine <i>M. tuberculosis</i>strains.</p

Antimycobacterial activity of the ion channel blockers against <i>M. tuberculosis</i>-infected macrophages.

<p> Effect of the inhibitors on the intracellular survival of <i>M</i>. <i>tuberculosis</i> (Mtb) within human monocyte-derived macrophages, three days post infection. Isoniazid (INH) was tested at 0.1 μg/ml, verapamil (VP), 10 μg/ml; thioridazine (TZ), 2.5 μg/ml; chlorpromazine (CPZ), 1.25 μg/ml; haloperidol (HAL), 1.25 μg/ml; flupenthixol (FPX), 1.25 μg/ml. The results are presented as a mean of the percentage of the survival ± SD. RIF, rifampicin; R, resistant; MDR, multidrug resistant; XDR, extensively drug resistant. The results were considered significant when *<i>P</i> < 0.05 and highly significant when **<i>P</i> < 0.01.</p

Effect of thioridazine on endocytic vesicles/phagosomes acidification.

<p> Confocal microscopy of human monocyte-derived macrophages infected with <i>M</i>. <i>bovis</i> BCG-GFP and stained with LysoTracker Red. A) to C): non-treated cells and (D) brightfield for non-treated cells; E) to G): cells treated with 2.5 μg/ml thioridazine (TZ) and (H) brightfield for cells treated with 2.5 μg/ml thioridazine. Comparing the non-treated cells with the treated ones, the LysoTracker staining is noticeable after macrophage treatment with the compounds. We can also observe co-localization of <i>M</i>. <i>bovis</i> BCG-GFP with LysoTracker Red. Data was analysed using a LSM 510 META laser scanning confocal microscope.</p

Mycobacterial intracellular ATP levels A) and viability B).

<p> M. tuberculosis H37Rv was exposed to the inhibitors (at 5X MIC) during three days. Bacterial viability was measured with MGIT960 system at day one, two, and three as described in Material and Methods. ATP was determined by using a luciferin-luciferase bioluminescence detection system at day one, two and three. Isoniazid (INH) and rifampicin (RIF) were used as controls. VP, verapamil; TZ, thioridazine; CPZ, chlorpromazine; FPX, flupenthixol; HAL, haloperidol. RLU, relative fluorescence units. The results were considered significant when *<i>P</i> < 0.05 and highly significant when **<i>P</i> < 0.01 and ***<i>P</i> <0.001.</p

Analysis of intracellular <i>M. tuberculosis</i>efflux pump gene expression.

<p> Bacterial efflux pump gene expression was analysed after the intracellular growth of <i>M</i>. <i>tuberculosis</i> strain 82/09 exposed to isoniazid at 0.1 μg/ml during three days. After this period, macrophages were lysed and RNA extracted from macrophage-grown bacilli using a GTC/Trizol based method. Relative expression of the five genes was determined by comparison of the relative quantity of the respective mRNA in the presence of isoniazid to the untreated control using the 2^-ΔΔCt method. Data was normalized to the <i>M</i>. <i>tuberculosis</i> 16S rDNA reference gene and presented as the mean fold change (±SD) compared with the control (non-treated macrophage-grown bacilli). A level of relative expression equal to 1 (black dotted line in the graph) indicates that the expression level was identical to the non-exposed condition. Genes showing expression levels equal or above one, when compared with the non-exposed condition, were considered to be overexpressed. The assays were performed in triplicate. Results were considered significant when *<i>P</i> < 0.05.</p