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

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

Accumulation of EtBr by the <i>M. tuberculosis</i> strains tested.

<p>The figure shows the accumulation of EtBr by the strains from adaptation process A as an example. The values at bold type correspond to the higher concentration of EtBr that cells can handle without detectable accumulation. The dotted line corresponds to the assay run using the EtBr concentrations for which influx-efflux are at equilibrium, in the presence of the EI verapamil, at sub-inhibitory concentrations. Panel (A): Parental strains (passage #0); Panel (B) strains after first passage with INH and Panel (C); strains after 26 passages with INH. INH: isoniazid.</p

Genotypic characterization of the strains and derived cultures exposed to isoniazid (adaptation process A).

<p><b>Legend:</b> INH: isoniazid; RIF: rifampicin; wt: wild type; Δ: deletion of <i>katG</i> gene; S: serine; L: leucine. MIRU-VNTR profile A: 2,4,2,2,3,4,2,3,2,3,2,4,2,2,6,1,6,3,1,3,1,7,2,2; profile B: 2,4,4,2,3,4,3,3,2,4,2,4,2,2,6,1,5,3,1,3,1,5,2,2; profile C: 2,1,4,2,1,3,2,3,2,2,2,5,1,2,6,1,6,3,3,3,2,4,2,2.</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

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