Thioridazine

Strains used include the wild type M. smegmatis mc²155 as well its transformants containing an overexpression plasmid pTA-M+, which overexpresses MtbTopI, or the control vector pKW-noI described previously^{23 (link)}. Cells were prepared by growing overnight at 37 °C in 7H9 media supplemented with 0.2% glycerol, 0.05% Tween 80, and 10% albumin, dextrose, sodium chloride (ADN). Overexpression strains were grown in the presence of 50 µg/ml hygromycin as well. The cells were grown to saturation and then diluted 1:100 in 7H9 without ADN supplementation. After another overnight growth to saturation, the cells were adjusted to OD₆₀₀ = 0.1 and diluted 1:5 in 7H9 media. Aliquots of 50 µL of the diluted cells (corresponding to ~10⁶ CFU) were then added to a clear-bottom 96-well plate that contained 50 µL of the serially diluted compound in the same media. The plate was incubated for 48 hours with shaking at 37 °C, and the optical density was measured approximately every 4 hours. The minimum inhibitory concentration is recorded as the concentration that prevented at least 90% growth when compared to the control wells. For some of the compounds, the MIC measurements were also carried out in the presence of the efflux pump inhibitor thioridazine (from Sigma Aldrich) at 6.25 µg/ml (half the MIC for growth inhibition by thioridazine alone). The experiments were replicated three times.

Mueller-Hinton broth was purchased from Sigma-Aldrich and Tween 80 from Merck-Schuchardt. The solvents used in this work were ethanol (>99.9%) from Panreac, toluene (>99.5%) from Riedel-de Häen, MTBE (>99.5%) from Fluka Analytical, and glycerol solution (86–89%) from Sigma-Aldrich. The antibiotics were levofloxacin and teicoplanin whilst the efflux pump inhibitors (EPIs) used were thioridazine and omeprazole, all from Sigma-Aldrich.

All cell lines were obtained from ATCC except SUM149 and SUM159 cells were obtained from the lab of Dr. Charles Perou, and SUM229 parental and EpCAM-sorted cells were obtained from the lab of Dr. Gary Johnson. SUM149, SUM159, SUM229, and EpCAM-sorted SUM149 and SUM229 cells were cultured in HuMEC medium (Gibco, Waltham, MA, USA) with supplements added and 5% FBS. MDA-MB-231 cells were cultured in DMEM (Gibco) with 10% FBS. All other cell lines were cultured in RPMI-1640 (Gibco) with 10% FBS. Penicillin/Streptomycin (Gibco) was added to all culture media. Cells were occasionally tested for mycoplasma with the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland). DRD2 antibody was obtained from Millipore (AB5084P) and used at 1:1000. Actin antibody was obtained from Cell Signaling Technologies. Thioridazine and quinpirole were obtained from Sigma-Aldrich, and SKF83959 was obtained from Cayman Chemical.