MCF7 breast cancer cell lines were purchased from the ATCC. Human immortalized fibroblasts (hTERT-BJ1) were originally purchased from Clontech, Inc. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% FBS (fetal bovine serum), 2 mM GlutaMAX, and 1% Pen-Strep in a 37°C humidified atmosphere containing 5% CO2, unless other-wise noted. Gibco-brand cell culture media (DMEM/F12) was purchased from Life Technologies. Bedaquiline was purchased from AdooQ Bioscience.
Bedaquiline
Manufactured by Adooq
Bedaquiline is a drug that inhibits the ATP synthase enzyme in Mycobacterium tuberculosis, the bacterium that causes tuberculosis. It is used as a part of a combination therapy for the treatment of multi-drug resistant tuberculosis.
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Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Mcf 7 breast cancer cell line, by American Type Culture Collection (1 mentions)
Htert bj1, by Takara Bio (1 mentions)
Prism 6, by GraphPad (1 mentions)
Spectramax m3, by Molecular Devices (1 mentions)
Rifampicin, by Merck Group (1 mentions)
Moxifloxacin, by Merck Group (1 mentions)
Tigecycline, by Adooq (1 mentions)
Clarithromycin, by Merck Group (1 mentions)
Rifabutin, by Merck Group (1 mentions)
Rifapentine, by Merck Group (1 mentions)
4 protocols using bedaquiline
1
Culturing MCF7 and hTERT-BJ1 Cells
2
Mycobacterial Growth Inhibition Assay
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The MIC values for taeanamide A and B against M. tuberculosis were determined using the REMA. For this purpose, the avirulent M. tuberculosis mc2 6230 strain was grown at 37 °C in Middlebrook 7H9 broth (Difco) supplemented with 10% ADS (50 g/L bovine albumin, 20 g/L dextrose, and 8.5 g/L NaCl), 0.2% glycerol, 24 μg/mL pantothenate, 0.2% casamino acids, and 0.05% Tween-80. For drug susceptibility testing, aliquots (100 μL) of the medium were added to all wells of a 96-well microtiter plate, and the final bacterial OD600 was adjusted to 0.005. Taeanamides A and B were added to each well using a 2-fold serial dilution method. Bedaquiline was purchased from Adooq Bioscience (#A12327) and was used as a reference compound. The plates were then incubated at 37 °C for 5 d. After this time, resazurin was added to each well (0.025% (w/v)), and the fluorescence value was read at 560–590 nm using a SpectraMax M3 multi-mode microplate reader (Molecular Devices, CA, USA). The concentrations required to inhibit bacterial growth by 50% (IC50) were determined by fitting the curves using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA).
3
Systematic Antibiotic Susceptibility Testing
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Fifteen longitudinal isolates, based on availability and regrowth, were sub-cultured into fresh MGIT tubes using 0.5 mL of the MGIT suspension (1:5 saline dilution), 0.1 mL of drug and 0.8 mL of oleic acid-albumin-dextrose-catalase (OADC). Most drug stock concentrations were purchased from MediaMage to obtain the following seven concentrations (mg/L) in MGIT (CCs recommended by WHO are bolded): INH (0.125, 0.25, 0.5, 1, 2, 4, 8), RIF (0.25, 0.5, 1, 2, 4, 8, 16), EMB (0.625, 1.25, 2.5, 5, 10, 20, 40), levofloxacin (LEVO) (0.125, 0.25, 0.5, 1, 2, 4, 8), moxifloxacin (MOXI) (0.062, 0.125, 0.25, 0.5, 1, 2, 4), LZD (0.125, 0.25, 0.5, 1, 2, 4, 8) and clofazimine (CFZ) (0.125, 0.25, 0.5, 1, 2, 4, 8).24 ,25 Bedaquiline was purchased from Adooq® (TMC-207, A12327) and nine concentrations were made up by MediaMage (0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16). A positive/growth control containing no drug, MGIT suspension (1:100 saline dilution) and 0.8 mL OADC, was used alongside each isolate. Details on loading of MGIT tubes and MIC interpretations are found in the supplementary material (Supplementary Methods
4
Antimicrobial Compounds for Research
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Clarithromycin (CLA), rifabutin (RFB), rifapentine (RFP), rifalazil (RFL), rifampicin (RIF), and moxifloxacin were purchased from Sigma-Aldrich (St. Louis, MO). Tigecycline (TGC) and bedaquiline (BDQ) were bought from Adooq Bioscience (Irvine, CA).
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