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Bafilomycin a

Manufactured by Merck Group
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Bafilomycin A is a laboratory reagent produced by Merck. It functions as a highly potent and specific inhibitor of vacuolar-type H+-ATPases (V-ATPases).

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Lab products found in correlation

Rapamycin, by Merck Group (13 mentions) Chloroquine, by Merck Group (10 mentions) Mg132, by Merck Group (10 mentions) Cycloheximide (chx), by Merck Group (9 mentions) 3 methyladenine, by Merck Group (7 mentions) Dimethyl sulfoxide (dmso), by Merck Group (7 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions) Tunicamycin, by Merck Group (6 mentions) Thapsigargin, by Merck Group (6 mentions) 3 methyladenine 3 ma, by Merck Group (6 mentions) Wortmannin, by Merck Group (5 mentions) N acetylcysteine, by Merck Group (5 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions) Pepstatin a, by Merck Group (4 mentions) Cytochalasin d, by Merck Group (4 mentions) Antimycin a, by Merck Group (4 mentions) Lysotracker red dnd 99, by Thermo Fisher Scientific (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Methyl β cyclodextrin, by Merck Group (3 mentions) Cisplatin, by Merck Group (3 mentions)

111 protocols using bafilomycin a

1

Cell Culture Drug Preparation Protocol

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Drugs for cell culture experiments were obtained as follows: Bafilomycin A (SML1661; Sigma-Aldrich), copanlisib (S2802; SelleckChem), leupeptin (L2884; Sigma-Aldrich), and ammonium chloride (A4514; Sigma-Aldrich).
Bafilomycin A was used at a concentration of 160 nM for all experiments. All other drug doses and incubation times are as indicated in the figure legends or directly on the figures. Bafilomycin A was dissolved in DMSO (D2650; Sigma-Aldrich). copanlisib, which is not DMSO soluble, was initially solubilized in a 10% trifluoroacetic acid (T6508; Sigma-Aldrich)/90% water solution to a concentration of 2 mM and then diluted with water to appropriate working stock concentrations. leupeptin (L2884; Sigma-Aldrich) was solubilized in sterile PBS.
For all drug treatments, the cells were maintained in a complete growth medium with serum, according to the recipes specified above.
Zhang K.K., Burns C.M., Skinner M.E., Lombard D.B., Miller R.A, & Endicott S.J. (2023). PTEN is both an activator and a substrate of chaperone-mediated autophagy. The Journal of Cell Biology, 222(9), e202208150.
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2

Chloroquine and Bafilomycin A1 in Drosophila

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Chloroquine (Sigma) diluted in PBS or PBS alone (vehicle control) was added to standard cornmeal-agar food for a final concentration of 1 mg/ml. Bafilomycin A1 dissolved in ethanol (Sigma) or ethanol along (vehicle control) was added to standard cornmeal-agar food to a final concentration of 400 nM. Crosses were set up in normal vials and transferred 24 hours later to Chloroquine, Bafilomycin A1, or the corresponding control vials. Third-instar larvae were collected, dissected, and processed for immunofluorescence.
Portela M., Yang L., Paul S., Li X., Veraksa A., Parsons L.M, & Richardson H.E. (2018). Lgl reduces endosomal vesicle acidification and Notch signaling by promoting the interaction between Vap33 and the V-ATPase complex. Science signaling, 11(533), eaar1976.
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3

Modulating Autophagy and MAPK in Embryos

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st24 embryos were dechorionated and incubated for 24 h in 50 nM Bafilomycin A (Sigma), 25 μM PD98059 (Cell Signaling), or 6 μM HA14‐1 (Sigma). Drugs were diluted in 3% DMSO/embryo medium. Medium was refreshed twice a day. Control embryos were grown in 3% DMSO/embryo medium.
Indrieri A., Carrella S., Romano A., Spaziano A., Marrocco E., Fernandez‐Vizarra E., Barbato S., Pizzo M., Ezhova Y., Golia F.M., Ciampi L., Tammaro R., Henao‐Mejia J., Williams A., Flavell R.A., De Leonibus E., Zeviani M., Surace E.M., Banfi S, & Franco B. (2019). miR‐181a/b downregulation exerts a protective action on mitochondrial disease models. EMBO Molecular Medicine, 11(5), e8734.
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4

Radiosensitivity Modulation in Cells

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High dose-rate γ-ray irradiation was performed using a Gammacell 40 Exactor (Nordion Inc., Kanata, Canada). The radioisotope source was 137Cs (132.2 TBq) and the dose rate was 0.9 Gy/min. Low dose-rate γ-ray irradiation was performed using a low dose and low dose-rate irradiation system (Sangyo Kagaku Co., Ltd., Tokyo, Japan). The radioisotope source was 137Cs (1.85 TBq), and the dose rate was 1 mGy/min [20 (link)]. During low dose-rate IR, the cells were maintained at 37°C in a humidified 5% CO2 incubator (normal cell culture condition), while high dose-rate IR was carried out in the atmosphere.
Pyocyanin is a mitochondrial ROS inducer [33 ]. Pyocyanin (Sigma) treatment (10 µM) was performed for 2 h. Bafilomycin A (BA), a specific inhibitor of the vacuolar type H (+)-ATPase in cells, inhibits the fusion of autophagosomes with lysosomes by inhibiting the acidification of organelles containing this enzyme (such as lysosomes and endosomes) [34 ]. BA (Cell Signaling Technology) treatment (100 nM) was performed for 46 h, including irradiation time. KU55933 (Sigma) is an ATM-kinase inhibitor. Cells were treated with 10 μM KU55933 for 46 h, including irradiation time.
Meng Q., Zaharieva E.K., Sasatani M, & Kobayashi J. (2021). Possible relationship between mitochondrial changes and oxidative stress under low dose-rate irradiation. Redox Report : Communications in Free Radical Research, 26(1), 160-169.
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5

Identifying Senescent Cells via β-Gal

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The ß-Gal enzyme was detected in the fluorescein isothiocyanate (FITC) spectrum as described before [64 (link)]. The cells were incubated with bafilomycin A (Sigma-Aldrich, St. Louis, MO, USA) 100 nM for 1 h to induce lysosomal alkalization. The population with smaller size, complexity, and lower β-Gal activity was determined as NS. For RS and SIPS, the opposite characteristics were considered since β-Gal activity brightness was previously reported in SC [34 (link),36 (link)].
Hernández-Mercado E., Prieto-Chávez J.L., Arriaga-Pizano L.A., Hernández-Gutierrez S., Mendlovic F., Königsberg M, & López-Díazguerrero N.E. (2021). Increased CD47 and MHC Class I Inhibitory Signals Expression in Senescent CD1 Primary Mouse Lung Fibroblasts. International Journal of Molecular Sciences, 22(19), 10215.
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6

Cell Line Treatment Protocols

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The HCT116 parental cell line and its p53-deficient variant (kindly provided by Professor Bert Vogelstein) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated FBS (Gibco) and PenStrep (100 U/ml penicillin, 100 μg/ml streptomycin; Sigma-Aldrich). Cell treatments with the antimetabolite 5-FU (Accord Healthcare Ltd), chloroquine (Sigma-Aldrich) or recombinant TRAIL (Thermo Fisher Scientific) were performed at concentrations and incubation times as indicated in the figures. In selected experiments the synthetic pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (zVAD-fmk; 10 μM; Peptide Institute Inc.), E64d (10 μM), pepstatin A (7 μM), 3-methyladenine (3-MA; 5 mM), bafilomycin A (Baf A; 100 nM), U18666A (5 ug/mL), brefeldin A (BFA; 100 nM), PUGNAc (25-100 μM) or methyl-β-cyclodextrin (MCD; 5 mM) (all from Sigma-Aldrich) were added to cell cultures 1 h prior to drug treatment. Benzyl-α-galNAc (2.5 mM; Sigma Aldrich) was added to cultures 56 h in advance of 5-FU treatment.
Akpinar B., Safarikova B., Laukova J., Debnath S., Vaculova A.H., Zhivotovsky B, & Olsson M. (2016). Aberrant DR5 transport through disruption of lysosomal function suggests a novel mechanism for receptor activation. Oncotarget, 7(36), 58286-58301.
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7

Measuring Calcium Influx in NHEKs

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[Ca2+]i was measured by a Fluo-4 NW Calcium Assay Kit (Life Technologies). Because the calcium ion concentration of the NHEK culture medium is low, we used low calcium contained-balanced salt solution (10 mM HEPES, pH7.4, 120 mM NaCl, 4 mM KCl, 1 mM KH2PO4, 1 mM MgCl2, 5 mM glucose, and 0.05 mM CaCl2) (Karvonen et al., 2000 (link)) instead of the Kit-included Assay buffer. To study calcium ion influx, cells were incubated with dye loading solution (balanced salt solution with Fluo-4 NW dye mix and 2.5 mM probenecid) at 37°C for 30 minutes, and then at room temperature for an additional 30 minutes. TIP39 peptide (5–25 μM), ionomycin (1 μM), thapsigargin (1 μM) (Enzo Life Science), bafilomycin A (0.5 μM) (Sigma-Aldrich), xestospongin C (5 μM), U73122 (5 μM), and W-7 (50 μM) (TOCRIS Bioscience, Bristol, UK) were added as indicated in dye loading solution, and then [Ca2+]i was monitored at an emission of 516 nm with excitation of 494 nm using a SpectraMax Gemini EM microplate Reader (Molecular Devices, Sunnyvale, CA) (Jiang et al., 2011 (link)). Fluo-4 signal changes (%) were defined as (F − F0)/F0 × 100. F was the target fluorescence signal intensity and F0 was the baseline calculated by averaging five time points just before the application of the stimulus.
Sato E., Muto J., Zhang L.J., Adase C.A., Sanford J.A., Takahashi T., Nakatsuji T., Usdin T.B, & Gallo R.L. (2016). The Parathyroid Hormone Second Receptor PTH2R and its Ligand Tuberoinfundibular Peptide of 39 Residues TIP39 Regulate Intracellular Calcium and Influence Keratinocyte Differentiation. The Journal of investigative dermatology, 136(7), 1449-1459.
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8

Analyzing TNF Production in BMDMs

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BMDMs were stimulated for 4 h with HkEc at a MOI of 20 plus either DMEM alone, or DMEM with 2.5 μM monensin (Sigma), 10 mM NH4Cl (Sigma), 100 μM chloroquine (Sigma), 2.5 μM brefeldin A (Merck) or 200 nM bafilomycin A (Sigma). Whole cell lysates were made and immunoblotted for TNF as described above.
Chew T.S., O'Shea N.R., Sewell G.W., Oehlers S.H., Mulvey C.M., Crosier P.S., Godovac-Zimmermann J., Bloom S.L., Smith A.M, & Segal A.W. (2015). Optineurin deficiency in mice contributes to impaired cytokine secretion and neutrophil recruitment in bacteria-driven colitis. Disease Models & Mechanisms, 8(8), 817-829.
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9

Bafilomycin A inhibition of autophagy

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U2OS cell samples were treated with 20 μM Bafilomycin A (Sigma) for 4 h. The Baf A stock was made at 1 mM in DMSO. Cell samples were then trypsinized, spun down for 3 min at 800g, and lysed in a 1% SDS buffer. Benzonase (0.25 μl) was added to each sample. The sample was left to incubate at RT for 10 min before heating at 60 °C for 5 min before performing a BCA assay to determine protein concentration. Ten microgram of each sample was loaded onto an Any kD precast gel (Bio-Rad) for optimal resolution followed by immunoblotting.
Amaya C., Cameron C.J., Devarkar S.C., Seager S.J., Gerstein M.B., Xiong Y, & Schlieker C. (2021). Nodal modulator (NOMO) is required to sustain endoplasmic reticulum morphology. The Journal of Biological Chemistry, 297(2), 100937.
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10

Investigating the Protective Role of SGLT2 Inhibitors in Cisplatin-Induced Cytotoxicity

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HK-2 cells (ATCC, Manassas, VA, USA), which were immortalized by transduction with human papillomavirus 16 E6/E7 genes, were maintained in a keratinocyte serum-free medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5 ng/mL human recombinant epidermal growth factor 1–53 and 50 μg/mL bovine pituitary extract at 37 °C in a humidified 5% CO2.
For analyzing the protective role of SGLT2 inhibitors in cisplatin-induced cytotoxicity, HK-2 cells were seeded in 6-well or 96-well plates and treated with 20 μM cisplatin (Sigma-Aldrich, St. Louis, MO, USA) in the absence or presence of 1–25 μM canagliflozin, dapagliflozin, or empagliflozin (APExBIO, Houston, TX, USA) for 24 h.
To test whether canagliflozin induces autophagy or modulates AMPK-mTOR pathway and whether the protective effect of canagliflozin on cisplatin-induced cytotoxicity occurs in autophagy or AMPK activation-dependent manner, the cells were seeded in 6-well or 96-well plates and treated with 20 μM cisplatin in the absence or presence of 10 μM canagliflozin and/or 10 μM chloroquine, 5 nM bafilomycin A (Sigma-Aldrich), or 1 μM compound C (APExBIO) for 24 h.
Park C.H., Lee B., Han M., Rhee W.J., Kwak M.S., Yoo T.H, & Shin J.S. (2022). Canagliflozin protects against cisplatin-induced acute kidney injury by AMPK-mediated autophagy in renal proximal tubular cells. Cell Death Discovery, 8, 12.
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