Bafilomycin

Manufactured by Enzo Life Sciences

Sourced in United States

Bafilomycin is a macrolide antibiotic that functions as a specific inhibitor of vacuolar-type H+-ATPases (V-ATPases). V-ATPases are proton pumps that play a crucial role in regulating intracellular pH and membrane trafficking within eukaryotic cells. As a potent and selective V-ATPase inhibitor, Bafilomycin is a valuable tool in biochemical and cell biology research.

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Lab products found in correlation

8 protocols using bafilomycin

Neuronal Lipid Metabolism and Signaling

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Primary cultures of hippocampal or cortical neurons were prepared from WT and ASM‐KO mouse embryos as described above. Where indicated AEA (Sigma, dissolved in ethanol) was added to the medium at 5–100 μM for 1 h, SM (Sigma, dissolved in ethanol) at 40 μM for 48 h, SMase (Sigma, dissolved in phosphate buffer saline) at 0.1 U/100 μl for 24 h, the CB₁ inhibitor SR141716 (Sigma, dissolved in DMSO) at 1 μM for 1 h, the NSM inhibitor GW4869 (Cayman Chemical, dissolved in DMSO) at 15 μM for 1 h, and the autophagy inhibitor bafilomycin (Enzo Life Sciences, dissolved in DMSO) at 0.1 μM for 24 h. In some instances, the following FAAH inhibitors were added for 1 h at a final concentration of 50 μM from stocks dissolved in DMSO: JNJ‐1661010 (Sigma), PF‐04557845 (MedChem), and URB597 (Selleckchem).
Primary skin fibroblasts were cultured in DMEM. PF‐04557845 (MedChem) was added to the medium for 1 h at final concentrations of 50 and 100 μM from stocks dissolved in DMSO.

Bartoll A., Toledano‐Zaragoza A., Casas J., Guzmán M., Schuchman E.H, & Ledesma M.D. (2020). Inhibition of fatty acid amide hydrolase prevents pathology in neurovisceral acid sphingomyelinase deficiency by rescuing defective endocannabinoid signaling. EMBO Molecular Medicine, 12(11), e11776.

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Senescence-associated beta-galactosidase assay

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After 48 hours the medium was removed from stretch or OS treated cells. Cells were treated with diluted bafilomycin (cat# BML-CM110-0100, Enzo Life Sciences, Farmingdale, NY, USA) and the negative control was treated with DMSO. Cells were incubated at 37°C for 1 hour. C12FDG (cat#7188 Setareh Biotech, Eugene, OR, USA) was added to the cells, except for the negative control. Cells were collected by adding trypsin, then trypsinization was stopped using complete medium. Cell were then transferred to a conical tube and centrifuged at 3,000 g for 10 minutes. The medium was removed and the pellet was re-suspended in 300 μL of annexin buffer treated with propidium iodine. The cells were run on a flow cytometer using a standard senescence-associated-β-galactosidase template (SA-β-Gal).

Richardson L.S., Radnaa E., Urrabaz-Garza R., Lavu N, & Menon R. (2020). Stretch, Scratch, and Stress: Suppressors and Supporters of Senescence in Human Fetal Membranes. Placenta, 99, 27-34.

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Compound Treatment Protocol for Cell Assays

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ML385 (Cayman Chemical, Ann Arbor, MI, USA) was dissolved in DMSO at a concentration of 10 mg/mL or 19.55 mM and added to the cells with media change at a concentration of 10 μM. Luperox^® TBHP 70% (Merck KGaA) was prediluted with ddH2O water and added to the cell media at the appropriate concentration. MG132 and Bafilomycin (both from Enzo Life Sciences, Lörrach, Germany) were dissolved in DMSO at a final concentration of 10 mM and 400 μM, respectively. For cell treatment, the compounds were administered directly to the culture media at 1:1000 dilution or up to a final concentration of 10 μM (MG132) or 400 nM (Bafilomycin). All compound treatments were performed for the indicated treatment time periods at 37 °C, 6% CO₂.

Semkova V., Haupt S., Segschneider M., Bell C., Ingelman-Sundberg M., Hajo M., Weykopf B., Muthukottiappan P., Till A, & Brüstle O. (2022). Dynamics of Metabolic Pathways and Stress Response Patterns during Human Neural Stem Cell Proliferation and Differentiation. Cells, 11(9), 1388.

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Synthesis and Characterization of Organometallic Compounds

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NiPT, PtPT, PdPT, T-AuPT, Au(PPh₃)PT, CuPT, and AgDT were synthesized in our laboratory and stored as a 10 mM stock solution in dimethyl sulfoxide (DMSO) at −20°C. Auranofin was purchased from Enzo Life Sciences International, Inc. (Plymouth Meeting, PA) and dissolved in DMSO as a 10 mM, stored at −20°C. Bortezomib (Cell Signaling Technology, Beverly, MA, USA) HA–ubiquitin–vinyl sulfone (HA-Ub-VS), and Ub-AMC (BostonBiochem, Cambridge, MA, USA). Antibodies were purchased from following sources: anti-AMPK, anti- phospho-AMPK, anti-mTOR, anti-phospho-mTOR, anti-P70S6K, anti-phospho-P70S6K, anti-S6, anti-phospho-S6, anti-ATF4, anti-CHOP, anti-phospho-eIF2a, anti-LC3, anti-USP14 (D8Q6S), and anti-PARP (Cell Signaling Technology, Beverly, MA, USA); anti-ubiquitin (P4D1) and anti-SQSTM1/P62(Santa Cruz Biotechnology, Santa Cruz, CA); anti-GAPDH and anti-HA-tag (Bioworld Technology, Nanjing, China); and anti-UCHL5 (Abcam, Cambridge, UK). Anti-phospho-USP14 and anti-phospho-UCHL5 were prepared by the Abgent, Inc. Bafilomycin, 3-MA, and E64D were purchased from Enzo Life Sciences (MA, USA). Lipofectamine 2000 (11668–027), goat anti-rabbit Alexa 488 (997773), and opti-MEM medium (22600050) were purchased from the Invitrogen^TM.

Chen J., Chen X., Xu D., Yang L., Yang Z., Yang Q., Yan D., Zhang P., Feng D, & Liu J. (2020). Autophagy Induced by Proteasomal DUB Inhibitor NiPT Restricts NiPT-Mediated Cancer Cell Death. Frontiers in Oncology, 10, 348.

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Cytotoxicity and Ion Signaling Assays

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Phorbol 12-myristate, 13-acetate (PMA) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and 1,25-dihydrixy-vitamin D3 from EMD Millipore (Billerica, MA, USA). The cytotoxicity assays CellTiter 96 (MTS assay) and CytoTox 96 [LDH (lactate dehydrogenase) assay] were purchased from Promega (Madison, WI, USA). Amphotericin B was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). ION Potassium Green-2 AM, a K⁺ indicator (Ex/Em 526/546), was purchased from Abcam (Cambridge, UK). LysoTracker Red DND-99 (Ex/Em 577/590), Lysosensor Yellow/Blue DND-160 (Ex/Em 329 and 380/440 and 540), and RPMI 1640 without phenol red were purchased from Thermo Forma/Fisher (Bothell, WA, USA). Gramicidin was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Bafilomycin and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3), Ex/Em 493/516) were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). L-

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-phosphatidylcholine (Egg-PC) and L-

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-phosphatidylethanolamine-N-lissamine rhodamine B sulfonyl (Rh-PE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA).

Ziglari T., Wang Z, & Holian A. (2021). Contribution of Particle-Induced Lysosomal Membrane Hyperpolarization to Lysosomal Membrane Permeabilization. International Journal of Molecular Sciences, 22(5), 2277.

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Assaying Cellular Senescence via Flow Cytometry

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Cells were plated in 6-well plates, with 300,000 cells per well (N = 3). The cells were left to attach for 12–24 h after the cells were plated, then they were treated with IL-6 at different concentrations. At the 48-h endpoint, the medium was removed. Cells were treated with diluted bafilomycin (cat# BML-CM110-0100, Enzo Life Sciences, Farmingdale, NY, United States) and the negative control was treated with DMSO. Cells were incubated at 37°C for 1 h. C12FDG (cat#7188 Setareh Biotech, Eugene, OR, United States) was added to the cells, except for the negative control. Cells were collected by adding trypsin, then trypsinization was stopped using complete medium. Cell were then transferred to a conical tube and centrifuged at 3,000 g for 10 min. The medium was removed and the pellet was re-suspended in 300 μL of annexin buffer treated with propidium iodine. The cells were run on a flow cytometer using a standard senescence-associated-β-galactosidase template (SA-β-Gal).

Omere C., Richardson L., Saade G.R., Bonney E.A., Kechichian T, & Menon R. (2020). Interleukin (IL)-6: A Friend or Foe of Pregnancy and Parturition? Evidence From Functional Studies in Fetal Membrane Cells. Frontiers in Physiology, 11, 891.

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Nigericin-Induced Autophagy Dynamics in HeLa Cells

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HeLa cells were plated onto coverslips 24 hr prior to 20 min nigericin treatment and recovery. At specified times post nigericin treatment, cells were fixed in 2 % paraformaldehyde, permeabilised with 0.1 % Triton X-100, blocked in PBS containing 2 % BSA and 5 % goat serum, and stained with anti-GalT (1:50, Sigma HPA010807) followed by AF594-conjugated goat anti-rabbit and DAPI. HeLa cells stably expressing mApple-Rab5 were plated onto coverslips 24 hr prior to treatment. Cells were left untreated, treated for 16 hr with bafilomycin (100 nM,Enzo Life Sciences, 100 μM stock in DMSO), or treated with nigericin for 20 min followed by a 60 min washout. Following treatment, cells were fixed for 10 min with methanol at –20 °C, blocked in PBS containing 2 % BSA and 5 % goat serum, and stained with anti-LC3b (1:400, Cell Signaling Technology #3868) followed by AF488-conjugated goat anti-rabbit. Coverslips were mounted onto glass slides with Fluoromount G (Southern Biotech) and sealed with nail polish. Following z-stack image acquisition, images were deconvolved using Huygens deconvolution online software tool.

Podinovskaia M., Prescianotto-Baschong C., Buser D.P, & Spang A. (2021). A novel live-cell imaging assay reveals regulation of endosome maturation. eLife, 10, e70982.

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Inflammasome Activation by Crystalline Silica

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BMDM were platted in a flat-bottom, tissue culture- treated 96-well plates at 1×105 cells/well in 100 μL of RPMI complete media and incubated with lipopolysaccharide (LPS) (20 ng/mL) for inflammasome priming. Cells were treated with 25 μM of hydroxychloroquine sulfate (Sigma-Aldrich cat. H0915-5MG), 25 μM imipramine hydrochloride (Sigma-Aldrich cat. 10899-5G), or 100 nM bafilomycin (EnzoLife Sciences cat. BML-CM110-0100) for 30 minutes prior to addition of cSiO₂. Cells were exposed to various doses (0-50 μg/mL) of cSiO₂ and plates were incubated in a 37°C water-jacketed CO₂ incubator (ThermoForma, Houston, TX) for 24 hours. Toxicity induced by cSiO₂ was determined by two complementary assays, a lactate dehydrogenase assay (Promega, cat. G1780) and a common colorimetric tetrazolium viability (MTS) assay (Promega, cat. G3580), and read on a plate reader (Molecular Devices SpectraMax M4 colorimetric microplate reader). In order to avoid artifacts in the optical density values, the MTS reaction was transferred to a clean plate to separate it from the cell/particle mixture adhered to the plate bottom. Data were normalized to a percent relative to the no particle, no HCQ control cells. NLRP3 inflammasome activation was assayed by measuring the release of IL-1β by using a commercially available ELISA kit (R&D Systems, cat. DY201).

Burmeister R., Rhoderick J.F, & Holian A. (2019). Prevention of Crystalline Silica-Induced Inflammation by the Anti-malarial Hydroxychloroquine. Inhalation toxicology, 31(7), 274-284.

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