Bafilomycin a1 bafa

Manufactured by Merck Group

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Bafilomycin A1 (BafA) is a laboratory reagent used in scientific research. It is a macrolide antibiotic that functions as a selective inhibitor of vacuolar-type H+-ATPase (V-ATPase), an enzyme responsible for the acidification of intracellular compartments. BafA is commonly employed in cell biology studies to investigate cellular processes dependent on pH regulation, such as endocytosis, autophagy, and intracellular trafficking.

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9 protocols using bafilomycin a1 bafa

Antimycin A-Induced Mitochondrial Dysfunction

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In the experiments, cells were seeded into 96-well plates (toxicity studies, mitochondrial membrane potential assessments), 12-well plates (Western blot analyses, toxicity studies, transmission electron microscopy samples, and caspase 3 activity measurements), or 8-well chamber slides (transfection studies, TUNEL staining, and confocal microscopy). Cell density was 15000 cells/well in 96-well plates, 20000 cells/well in 8-well chamber slides, and 200000 cells/well on 12-well plates. ARPE-19 cells were cultured in tissue culture-treated plates, while hRPE cells were plated on well surfaces coated with Matrigel (Corning, Corning, NY, USA). The cells were incubated for 72-96 hours until fully confluent, unless otherwise stated, and were treated with varying concentrations of antimycin A (Aa) (Sigma-Aldrich, St. Louis, MO, USA), with or without pretreatment with 30 μM chloroquine (CQ) (Sigma-Aldrich, St. Louis, MO, USA) or 50 nM bafilomycin A1 (BafA) (Sigma-Aldrich, St. Louis, MO, USA) in serum-free culture medium. Cells and medium were collected after the indicated times, specific for each analysis, and analyzed according to the protocols of the different methods.

Hytti M., Korhonen E., Hyttinen J.M., Roehrich H., Kaarniranta K., Ferrington D.A, & Kauppinen A. (2019). Antimycin A-Induced Mitochondrial Damage Causes Human RPE Cell Death despite Activation of Autophagy. Oxidative Medicine and Cellular Longevity, 2019, 1583656.

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Autophagy Inhibitors Modulate Cell Responses

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24 h prior to in vitro experiment, cells were passaged and plated in 24-well plates at a density of 5 × 10⁴ cells per well. Specified cells were pre-treated with LPS (10 ng/ml; Sigma, L2630), 3 h prior to drug treatment. The following autophagy inhibitors were used: Bafilomycin A₁ (BafA) (20 nM; Sigma-Aldrich, B1793), 3-MA (100 μM; Sigma-Aldrich, M9281) and MRT68921 (10 µM; Sigma-Aldrich, SML1644). Treatment with the inhibitors was carried out for 6 h, following which all samples were lysed in Laemmli buffer and processed for western blotting. For all in vitro experiments, n = 3 replicates/group with 3 independent experiments performed.

Hegdekar N., Sarkar C., Bustos S., Ritzel R.M., Hanscom M., Ravishankar P., Philkana D., Wu J., Loane D.J, & Lipinski M.M. (2023). Inhibition of autophagy in microglia and macrophages exacerbates innate immune responses and worsens brain injury outcomes. Autophagy, 19(7), 2026-2044.

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Autophagy and Apoptosis Modulation in Cancer Cells

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Specific antibodies against light chain 3 (LC3), Beclin 1, p62, B-cell lymphoma 2 (Bcl-2), poly ADP-ribose polymerase (PARP), caspase-3, mammalian target of rapamycin (mTOR), phosphorylated mammalian target of rapamycin (p-mTOR), and phosphorylated protein kinase B were purchased from Cell signaling Technologies, Inc. (Danvers, MA). Antireduced glyceraldehyde-phosphate dehydrogenase (GAPDH) monoclonal antibody was purchased from Sigma (St Louis, MO). Bortezomib (BZ) was purchased from Santa Cruz, doxorubicin (Dox) and dexamethasone (Dex) were from Sigma, bafilomycin A1 (BafA) and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich. Chemoluminescence detection system and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Beyotime Institute of Biotechnology, Nantong, People’s Republic of China.

Shi M., Cheng L., Zhang Z., Liu Z, & Mao X. (2014). Ferroferric oxide nanoparticles induce prosurvival autophagy in human blood cells by modulating the Beclin 1/Bcl-2/VPS34 complex. International Journal of Nanomedicine, 10, 207-216.

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Quantification of Autophagy in Mouse Embryonic Fibroblasts

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Mouse embryonic fibroblasts (MEFs) were plated onto glass coverslips and were subsequently transfected with the indicated plasmids for 36 h. The cells were then left untreated, incubated in starvation medium for 2 h, or in medium containing 50 nM bafilomycin A₁ (BafA, Sigma) or 200 nM rapamycin (Sigma) for 2 h. Longer incubations were found to compromise the survival of Stk3^+/−;Stk4^−/− MEFs (data not shown). Cells were fixed in 4% paraformaldehyde in PBS for 10 min, permeabilized in 0.2% Triton X-100 in PBS for 10 min, and then processed according to Cell Signaling immunofluorescence protocol (www.cellsignal.com). Images were acquired by confocal microscopy and LC3 puncta were quantified using IMARIS spot-counting software.

Wilkinson D.S., Jariwala J.S., Anderson E., Mitra K., Meisenhelder J., Chang J.T., Ideker T., Hunter T., Nizet V., Dillin A, & Hansen M. (2014). Phosphorylation of LC3 by the Hippo kinases STK3/STK4 is essential for autophagy. Molecular cell, 57(1), 55-68.

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Modulating Dendritic Cell Responses to Bacterial Pathogen

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DCs (2·10⁶ cells/ml) were stimulated with UV-treated USA300 at MOI 10, unless otherwise stated, and incubated at 37°C in 5% CO₂ at indicated hours. DCs were treated with 100 µg/ml mannan from Saccharomyces cerevisiae (Sigma-Aldrich, St. Louis, MO, USA) 30 min prior to bacteria stimulation. Prior to bacterial stimulation BMDCs were pre-treated with 0.5 µg/ml cytochalasin D (CytD) (Sigma-Aldrich) for 1 h to inhibit bacterial uptake, with 300 µM apocynin (R&D systems, Minneapolis, MN, USA) for 30 min to inhibit assembly of the NADPH oxidase and ROS production and with 50 nM bafilomycin A1 (BafA) (Sigma-Aldrich) or 40 mM NH₄Cl (Sigma-Aldrich) for 1 h to inhibit phagosome acidification. MAPK pathways were inhibited in DCs by pre-treating for 1 h with 25 µM JNK inhibitor (Sigma-Aldrich, SP600125), 10 µM p38 inhibitor (Sigma-Aldrich, SB203580) or 10 µM MEK 1/2 inhibitor (Sigma-Aldrich, UO126). Factors present during inflammation such as GM-CSF (15 ng/ml) or IFN-γ (1 ng/ml, Thermo Fisher Scientific) was added to the medium of DCs just prior to bacterial stimulation.

Eld H.M., Johnsen P.R., Nielsen E.M., Jørgensen F.Z., Lindstrøm-Svendsen M., Baldry M., Ingmer H, & Frøkiær H. (2022). Soluble C-Type Lectin-Receptor Ligands Stimulate ROS Production in Dendritic Cells and Potentiate Killing of MRSA as Well as the MRSA Induced IL-12 Production. Frontiers in Immunology, 13, 845881.

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Growth Factor Activation Pathway Protocol

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EGF was purchased from Invitrogen, Carlsbad, CA, USA. AREG and TGF-

α

were from R&D Systems, Minneapolis, MN, USA. If not otherwise indicated, 10 nM of EGF and 20 nM AREG or TGF-

α

were used. EGFR inhibitor AG-1478 (Sigma-Aldrich, Saint Louis, MO, USA) was used at a concentration of 1

μ

M. The protein synthesis inhibitor cycloheximide (CHX, Sigma-Aldrich, USA) was used at a concentration of 35.5

μ

M. The vacuolar V-ATPase inhibitor bafilomycin A

_{1}

(BafA, Sigma-Aldrich, USA) was used at a concentration of 100 nM.

Kamentseva R.S., Kharchenko M.V., Gabdrahmanova G.V., Kotov M.A., Kosheverova V.V, & Kornilova E.S. (2023). EGF, TGF-α and Amphiregulin Differently Regulate Endometrium-Derived Mesenchymal Stromal/Stem Cells. International Journal of Molecular Sciences, 24(17), 13408.

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Autophagy Modulation in Microfluidic Devices

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Bafilomycin A1 (BafA; 100 nM; Sigma) and 50 μM CQ (Enzo Life Sciences) were prepared in culture media from a dimethyl sulfoxide (DMSO)-based stock solution. 3-MA (5 mM; Sigma) was dissolved directly in media. Small molecules were supplied to the microfluidic devices continuously for the duration of the experiments. Control cells were treated with the DMSO vehicle.

Kim S.W., Ehrman J., Ahn M.R., Kondo J., Lopez A.A., Oh Y.S., Kim X.H., Crawley S.W., Goldenring J.R., Tyska M.J., Rericha E.C, & Lau K.S. (2017). Shear stress induces noncanonical autophagy in intestinal epithelial monolayers. Molecular Biology of the Cell, 28(22), 3043-3056.

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Modulating Autophagy in Cell Lines

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Cells were reverse transfected with 10 nM of non-silencing control or specific gene-targeting siRNAs (siNS, siFYCO1, or siSTK4; Dharmacon) using Opti-MEM and lipofectamine RNAiMAX according to the supplier’s recommendations (Life Technologies). After 48 h, cells were collected and used for experiments. Transient plasmid transfections were performed using Opti-MEM and Lipofectamine 2000 (Life Technologies), and cells were collected for experiments 24 h after transfection. For experiments with cells expressing siRNAs and plasmids, cells were transfected with siRNAs for 24 h and then transfected with the plasmid of interest for an additional 24 h before analysis.
For autophagy flux assays, cells were washed three times with normal medium, normal medium containing the late-autophagy blocking compound bafilomycin A₁, (BafA, Sigma) at 50 nM, or starvation medium (Earle’s Balanced Salt Solution (Life Technologies) before incubation in the same media for the indicated times at 37°C.

Nieto-Torres J.L., Shanahan S.L., Chassefeyre R., Chaiamarit T., Zaretski S., Landeras-Bueno S., Verhelle A., Encalada S.E, & Hansen M. (2021). LC3B phosphorylation regulates FYCO1 binding and directional transport of autophagosomes. Current biology : CB, 31(15), 3440-3449.e7.

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Mechanistic Insights into Autophagy Modulation

Cited 1 time

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Transfections were done using Lipofectamine 3000 (Thermo Fisher, L3000008) for plasmid DNA. Doxorubicin (Sigma-Aldrich, D1515) was diluted in water to 5 mM and used at 1 μM for 48 h. Bafilomycin A₁ (BafA; Sigma-Aldrich, B1793) was diluted in DMSO to 200 μM and used at 100 nM for 4 h. Chloroquine (CQ; Sigma-Aldrich, C6628) was diluted in DMSO to 50 mM and used at 20 μM for 4 h. Rapamycin (Sigma-Aldrich, R0395) was diluted in ethanol to 2 mM and used at 200 nM for 4 h. Leu-Leu methyl ester hydrobromide (LLOMe; Sigma-Aldrich, L7393) was diluted in DMSO to 1 M and used from 0.001 to 1.4 mM for 6 h. Sorafenib tosylate (MedChemExpress, HY-10201A) was used from 5 to 14 μM for 24 h in vitro and 100 mg/kg in vivo daily. FITC-dextran (Sigma-Aldrich, FD40S) was directly diluted in the medium to 0.1 mg/mL. MTT (Thermo Fisher, M6494) was diluted in PBS (137 mM NaCl, 2.7 mM KCl, 12.5 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) to 5 mg/mL. For high starvation, cells were washed twice with PBS and starved in low glucose DMEM lacking amino acids (USBiological, D9800-13) and serum. To overexpress the TFEB variant with pronounced nuclear localization characteristics [45 (link)] the plasmid pCIP-caTfeb (Addgene, 79,013; deposited by Reuben Shaw) was transfected into Huh-7 VTRNA1-1 KO cells.

Ferro I., Gavini J., Gallo S., Bracher L., Landolfo M., Candinas D., Stroka D.M, & Polacek N. (2021). The human vault RNA enhances tumorigenesis and chemoresistance through the lysosome in hepatocellular carcinoma. Autophagy, 18(1), 191-203.

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