Gentamicin sulfate

DMEM F12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), HBSS (Hank's Balanced Salt Solution), FBS (fetal bovine serum), ultraglutamine 1, and gentamicin sulfate were purchased from Lonza (Basel, Switzerland). Accutase™ Cell Detachment and FITC Annexin V Apoptosis Detection Kit were obtained from BD Biosciences (Franklin Lakes, New Jersey, USA). TrypLE™ Express was from Gibco (Waltham, MA, USA). MitoPy1 [4-[4-[3-Oxo-6′-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)spiro [isobenzofuran-1(3H), 9′-[9H]xanthen]-3′-yl]-1-piperazinyl]butyl]triphenyl-phosphonium iodide) was from Tocris Bioscience (Bristol, United Kingdom). Bovine serum albumin (BSA), DCF-DA (2,7-dichlorofluorescin diacetate), NAC (N-Acetyl-L-cysteine), DMSO (Dimethyl Sulfoxide), paraformaldehyde (PFA), propidium iodide, and DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol 96% was from Chempur. Celastrol, with purity more than 98%, was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Phospho-histone H2A.X (Ser139) monoclonal antibody (CR55T33) Alexa Fluor 488 eBioscience™ was obtained from Invitrogen (Carlsbad, CA, USA).

Acetonitrile, methanol, formic acid, DPPH (1,1-diphenyl-2 picrylhydrazyl radical), ABTS (2,2-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) radical cation), TPTZ (2,4,6-tri(2-pyridyl)-s-triazine), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), FeCL₃, acetic acid, ganoderic acid A, caffeic acid, and resveratrol were purchased from Sigma-Aldrich (Steinheim, Germany). Apigenin 7-O-glucoside, isorhamnetin 3-O-glucoside, kaempferol 3-O-glucoside, quercetin 3-O-glucoside, chlorogenic, p-coumaric, quinic, ferulic and rosmarinic acids, procyanidin B2, and (-)-epicatechin were purchased from Extrasynthese (Lyon, France). All reagents were of analytical grade. DMEM/F12 (Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12), DMEM (Dulbecco’s Modified Eagle’s Medium), HBSS (Hank’s Balanced Salt Solution), FBS (fetal bovine serum), UltraGlutamine 1, and gentamicin sulfate were purchased from Lonza (Basel, Switzerland). TrypLE Express and MEM (Minimum Essential Media) were from Gibco (Waltham, MA, USA). MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and DMSO (dimethyl sulfoxide) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Isopropanol was purchased from Chempur (Piekary Śląskie, Poland).

HEK293-T cells were obtained from ATCC. B16F10 and DC2.4 cells were gifts from the Irvine lab. B16.SIY cells were a gift from the Spranger lab. MC-38 and TC-1 cells were gifts from the Wittrup lab. KP lines were gifts from the Jacks lab. CD8⁺ 58^-/-, Sf9, and Hi5 cells were gifts from the Garcia lab.
HEK 293-T, B16F10, B16.SIY, and MC-38 cells were cultured in Dulbecco’s Modified Eagle’s Medium (ATCC) supplemented with 10% fetal bovine serum (FBS, R&D Systems), 100 U/mL penicillin (Thermo), and 100 μg/mL streptomycin (Thermo). TC-1, DC2.4, and 58^-/^- lines were cultured in RPMI-1640 media (ATCC) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All mammalian cell lines and assay cultures were maintained at 37°C and 5% CO₂. All cells were frequently tested and confirmed negative for mycoplasma contamination. B16F10 cells used for tumor challenge studies also tested negative for rodent pathogens.
Sf9 cells were cultured in Sf-900 III SFM (Gibco) supplemented with L-glutamine, 10% FBS (R&D Systems), and 20 μg/mL gentamicin sulfate (Lonza). Hi5 cells were cultured in Insect-XPRESS (Lonza) supplemented with 10 μg/mL gentamycin sulfate (Lonza). Insect cell lines were maintained at 27°C with shaking at 120 rpm.

Human bone marrow derived-mesenchymal stem cells (hBM-MSC) were purchased from Lonza (Walkersville, MD) and maintained in MSC basal media supplemented with L-glutamine, gentamicin sulfate, amphotericin and mesenchymal cell growth supplement (Lonza; Walkersville, MD). After the third passage, the cells were cultured in vesicle-depleted media. Vesicle depletion from media was performed by tangential flow filtration (TFF) using a sterile 500 kDa molecular weight cut off MidiKros filter lined with a modified polyethersulfone membrane (Repligen; Waltham, MA). The permeate, containing the vesicle-depleted MSC media, was collected and passed through a 0.22 μm filter before storing at 4°C. Human hepatocytes (HH; Sciencell, United Kingdom) and PLC cells (ATCC; Manassas, VA) were cultured in untreated plates. KMBC (provided by Dr. Gregory Gores, Mayo Clinic), HepG2 and Hep3B (ATCC; Manassas, VA) cells were cultured in collagen-coated plates. The aforementioned cells were maintained in Dulbecco’s modified eagle media (DMEM) high glucose media supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). HL-60 promyeloblasts (ATCC; Manassas, VA) were cultured with Iscove’s modified Dulbecco’s medium supplemented with 20% FBS in T75 flasks. RAW264.7 murine macrophages (ATCC; Manassas, VA) were cultured with DMEM high glucose media supplemented with 10% FBS.

One day before infection, mycobacterial cultures were diluted to a density corresponding with early log-phase growth (optical density at 600 nm (OD₆₀₀) of 0.4). Stm was grown either in LB broth or on LB agar with appropriate antibiotics. After overnight incubation, Stm liquid cultures were diluted 1:33 and cultured for an additional 3–4 h, while plate-grown Stm was suspended in PBS by rinsing the agar plates. Bacterial density was determined by measuring the OD₆₀₀ and the bacterial suspension was diluted in cell culture medium without antibiotics to reach a multiplicity of infection (MOI) of 10 (unless indicated otherwise). Accuracy of bacterial density measurements was verified by a standard colony-forming unit (CFU) assay. Cell cultures (HeLa for Stm infections and MelJuSo for Mtb infections), seeded in 96-well flat-bottom plates as described below, were inoculated with 100 μl of the bacterial suspension, centrifuged for 3 min at 800 rpm, and incubated at 37 °C/5% CO₂ for 20 min if infected with Stm or 60 min if infected with Mtb. The plates were then washed with culture medium containing 30 μg/ml gentamicin sulfate (Lonza BioWhittaker, Basel, Switzerland) and incubated at 37 °C and 5% CO₂ in a medium containing 5 μg/ml gentamicin and the indicated chemical compounds until readout by flow cytometry or CFU, as indicated.

Tamoxifen citrate (tamoxifen) and rifampicin were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands). Isoniazid was purchased from SelleckChem (Munich, Germany). Gentamicin sulfate was bought from Lonza BioWhittaker (Basel, Switzerland), and hygromycin B was acquired from Life Technologies-Invitrogen (Bleiswijk, The Netherlands). Fulvestrant, 17β-estradiol, and Hoechst 33342 were purchased from Sigma-Aldrich. Rabbit polyclonal anti-TFEB (RRID:AB_11220225) was purhcased from Cell Signaling Technology (Leiden, The Netherlands). Phalloidin-iFluor 405 was obtained from Abcam (Cambridge, UK). Goat anti-rabbit IgG (H+L) AlexaFluor647 conjugate (RRID:AB_2536101) was purchased from Thermo Fisher Scientific (Breda, The Netherlands). All compounds, except Gentamicin sulfate and hygromycin B, were dissolved in 100% dimethyl sulfoxide (DMSO; Sigma-Aldrich) in stock concentrations of 10 mM, aliquoted, and kept at −80°C.