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Valinomycin

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Valinomycin is a cyclic peptide antibiotic produced by certain Streptomyces bacteria. It functions as a potassium ionophore, facilitating the transport of potassium ions across cell membranes.

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Lab products found in correlation

Antimycin, by Merck Group (3 mentions) Nigericin, by Bio-Techne (2 mentions) Fluorescent labeled secondary antibody, by Thermo Fisher Scientific (2 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions) Bafilomycin a1, by Merck Group (2 mentions) Ru360, by Merck Group (2 mentions) Fk506, by Cayman Chemical (2 mentions) Anti tom20, by Proteintech (2 mentions) Oligomycin, by Cayman Chemical (2 mentions) Actinonin, by Cayman Chemical (2 mentions) 1 na pp1, by Cayman Chemical (2 mentions) Anti pdh, by Abcam (2 mentions) Hydrogen peroxide solution, by Merck Group (1 mentions) Bcecf am, by Thermo Fisher Scientific (1 mentions) Euk 134, by Cayman Chemical (1 mentions) Px 12, by Bio-Techne (1 mentions) Rapamycin, by Merck Group (1 mentions) Ammonium chloride, by Merck Group (1 mentions) Choline oxidase, by Avantor (1 mentions) Amplex red 10 acetyl 3 7 dihydroxyphenoxazine, by Cayman Chemical (1 mentions)

8 protocols using valinomycin

1

Investigating Cell Signaling Modulators

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Reagents and salts for buffer preparation were purchased from Sigma-Aldrich (MO, USA). Lipofectamine® 2000, media culture and supplements were acquired from Life Technologies (NY, USA). EUK-134 was purchased from Cayman Chemical Company (MI, USA). PX-12, nigericin, and valinomycin were acquired from Tocris (MO, USA). BCECF-AM was acquired from Thermo Fisher Scientific (MA, USA). The 30% hydrogen peroxide solution used in this work was acquired from Merck (Darmstadt, Germany).
Hernández H., Parra A., Tobar N., Molina J., Kallens V., Hidalgo M., Varela D., Martínez J, & Porras O. (2018). Insights into the HyPer biosensor as molecular tool for monitoring cellular antioxidant capacity. Redox Biology, 16, 199-208.
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2

Mitochondrial Function Modulation in Cell Lines

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HEK293T cells were obtained from ATCC. HeLa cells were a gifted by Sabrina Spencer. Beige fat cells were a gift from Shingo Kajimura. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (Invitrogen), penicillin, streptomycin (100units/mL), and 1 mM L-glutamine. Cells were treated at indicated concentrations with CCCP (SigmaAldrich), valinomycin (Tocris), Antimycin (SigmaAldrich) Oligomycin (Cayman Chemical), Actinonin (Cayman Chemical), FK506 (Cayman Chemical), 1-NA-PP1 (Cayman Chemical), Bafilomycin A1 (SigmaAldrich), and Ru360 (EMDMillipore). Antibodies were purchased from the following providers: anti-PDH, Abcam; anti-Tom20, Proteintech. Fluorescent labeled secondary antibodies were purchased from ThermoFisher.
Ramirez A., Old W., Selwood D.L, & Liu X. (2021). Cannabidiol activates PINK1-Parkin-dependent mitophagy and mitochondrial-derived vesicles. European journal of cell biology, 101(1), 151185.
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3

Fluorescent Dye Assay Reagents

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Dipicrylamine sodium salt was obtained from Biotium Inc. (Fremont, CA). Rapamycin, dimethyl sulfoxide (DMSO), and ammonium chloride were obtained from Sigma-Aldrich. Valinomycin and nigericin were obtained from Tocris Bioscience (Bristol, UK). Standard salts used for solutions were obtained from Merck.
Matamala E., Castillo C., Vivar J.P., Rojas P.A, & Brauchi S.E. (2021). Imaging the electrical activity of organelles in living cells. Communications Biology, 4, 389.
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4

Mitochondrial Function Modulation in Cell Lines

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HEK293T cells were obtained from ATCC. HeLa cells were a gifted by Sabrina Spencer. Beige fat cells were a gift from Shingo Kajimura. Cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum (Invitrogen), penicillin, streptomycin (100units/mL), and 1 mM L-glutamine. Cells were treated at indicated concentrations with CCCP (SigmaAldrich), valinomycin (Tocris), Antimycin (SigmaAldrich) Oligomycin (Cayman Chemical), Actinonin (Cayman Chemical), FK506 (Cayman Chemical), 1-NA-PP1 (Cayman Chemical), Bafilomycin A1 (SigmaAldrich), and Ru360 (EMDMillipore). Antibodies were purchased from the following providers: anti-PDH, Abcam; anti-Tom20, Proteintech. Fluorescent labeled secondary antibodies were purchased from ThermoFisher.
Ramirez A., Old W., Selwood D.L, & Liu X. (2021). Cannabidiol activates PINK1-Parkin-dependent mitophagy and mitochondrial-derived vesicles. European journal of cell biology, 101(1), 151185.
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5

Lipid Substrate Assay for Phospholipase D

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Bacterial (Streptomyces chromofuscus), peanut, and cabbage PLD was purchased from Enzo and sigma-aldrich respectively. Amplex Red 10-Acetyl-3,7-dihydroxyphenoxazine (Cayman chemical). Horseradish peroxidase (VWR), choline oxidase (VWR). All lipids including 1,2-dioctanoyl-sn-glycero-3-phosphocholine (C8-PC), 1-palmitoyl-2-(dipyrrometheneboron difluoride) undecanoyl-sn-glycero-3-phosphocholine (TopFluorPC), 1-(dipyrrometheneboron difluoride) undecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (TopFluorLPC), 1 -palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC or 18:1 PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol (POPEtOH), and 1,2-dioleoyl-sn-glycero-3-phosphoethanol (DOPEtOH) were purchased from Avanti Polar Lipids (Alabaster, AL) in chloroform at stock concentrations of 10 or 25 mg/mL except C8-PC and TopFluor lipids which were purchased as powder. Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) was from Tocris Bioscience and made at a stock concentration of 1mM in ethanol. Valinomycin was also purchased from Tocris Bioscience and made at a stock concentration of 200μM in ethanol. The detergent n-dodecyl-β-D-maltoside (DDM) was purchased from Anatrace (D310S) and made at 200mM. ACMA (9-amino-6-chloro-2-methoxyacridine), was purchased from Life Technologies and made at a stock concentration of 2mM in ethanol.
Chung H.W., Petersen E.N., Cabanos C., Murphy K.R., Arif Pavel M., Hansen A.S., Ja W.W, & Hansen S.B. (2018). A Molecular Target for an Alcohol Chain Length Cutoff.. Journal of molecular biology, 431(2), 196-209.
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6

Mitochondrial Membrane Potential Analysis

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Mitochondrial membrane potential was measured using the JC-1 dye (Merck). HuCCT-1, OCUG-1 and TFK-1 cells were seeded in transparent 96-well microplates using a seeding number of 10,000 cells per well and let grown for 24 hours. Cells were washed with serum-free DMEM and incubated with 1 μM, 0.1 μM and 0.01 μM ouabain for 6 hours. Prior to staining, JC-1 was warmed in a water bath, vortexed and centrifuged at 17,000 g for 10 minutes as described [38 (link)]. After incubation, cells were washed with serum-free media and 5 μM staining solution in Hanks’ Balanced Salt Solution (HBSS; ThermoFisher Scientific, Waltham, MA, USA) were added. The positive control Valinomycin (Tocris, Bristol, United Kingdom) was added with the JC-1 dye to avoid prolonged incubation and cytotoxicity. For staining, cells were incubated for 30 minutes in a cell incubator. Then, cells were washed twice with HBSS and fluorescence was measured at excitation 485 nm / emission 525 nm (green) and excitation 480 nm / emission 590 nm (red) using a Spark multimode reader. Changes of mitochondrial potential were calculated as ratio of red to green fluorescence.
Mayr C., Kiesslich T., Bekric D., Beyreis M., Kittl M., Ablinger C., Neureiter E., Pichler M., Prinz F., Ritter M., Neureiter D., Jakab M, & Dobias H. (2023). Ouabain at nanomolar concentrations is cytotoxic for biliary tract cancer cells. PLOS ONE, 18(6), e0287769.
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7

Mitochondrial Membrane Potential Assessment

Cited 1 time
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Cells were treated with CCCP (Sigma-Aldrich, C2759, 20 μM unless stated otherwise), CDDO (MedChemExpress, HY-14909, 2.5 μM), tunicamycin (MedChemExpress, HY-A0098, 10 μM), GTPP (GTPP hexafluorophosphate, MedChemExpress, HY-102007A, 10 μM), oligomycin (CST, 9996L, 10 μM or 25 μM), antimycin (Sigma-Aldrich, A8674, 50 μM), valinomycin (BioTechne, 3373, 0.5 μM), pepstatin A (Enzo, ALX-260-085-M005, 50 μM), 1,10-phenanthroline monohydrate (Sigma-Aldrich, P9375, 500 μM), E-64 (Sigma-Aldrich, E3132, 1 μM), E-64d (Enzo, BML-PI107-0001, 50 μM), zVAD (Z-VADFMK, PeptaNova, 3188-v, 20 μM), 3,4-dichloroisocoumarin (Sigma-Aldrich, D7910, 100 μM), Pefabloc SC Plus (Sigma-Aldrich, 1187360100, 500 μM), ISRIB (Sigma-Aldrich, SML0843, 200 nM), haemin (Sigma-Aldrich, 51280, 20 μM) or cycloheximide (Sigma-Aldrich, C4859, 20 μg ml−1) for the indicated times.
Where indicated, cells were transfected 24 to 36 h before treatment using polyethylenimine (PEI 25000, Polysciences) or Turbofectin (OriGene Technologies). Transductions were performed as described previously26 (link).
Mitochondrial membrane potential was assessed using 100 nM tetramethylrhodamine (TMRM; Thermo Fisher Scientific) following the manufacturer’s instructions.
Fessler E., Eckl E.M., Schmitt S., Mancilla I.A., Meyer-Bender M.F., Hanf M., Philippou-Massier J., Krebs S., Zischka H, & Jae L.T. (2020). A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol. Nature, 579(7799), 433-437.
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8

Measuring CFTR, ENaC, and BK Channel Activities

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HBEC were mounted in Ussing chambers connected to a VCC MC6 or MC8 voltage clamp unit (Physiological Instruments, San Diego, CA, USA). CFTR activity was measured as the change in short-circuit current (ISC) caused by CFTR inhibition with 10 µM CFTRinh-172 (#C2992; MilliporeSigma, Burlington, MA, USA) after CFTR stimulation with 10 µM forskolin (#F3197; MilliporeSigma) in the presence of 10 µM amiloride (#A7410; MilliporeSigma) under a basolateral-to-apical Cl− gradient. ENaC activity was measured as the change in ISC after amiloride. For measurements of BK channel function, the basolateral membrane of HBEC mounted in Ussing chambers was permeabilized with 20 µM Amphotericin B (#A2411; MilliporeSigma), 10 µM Nigericin (#4312; Bio-Techne Corporation, Minneapolis, MN, USA), and 10 µM Valinomycin (#3373; Bio-Techne Corporation). BK channel activity was then measured as the change in ISC following stimulation with 10 µM ATP (#A9187; MilliporeSigma) in the presence of 10 µM amiloride under a basolateral-to-apical K+ gradient and an apical-to-basolateral Na+ gradient as previously described24 (link).
Kim M.D., Chung S., Baumlin N., Qian J., Montgomery R.N., Sabater J., Berkland C, & Salathe M. (2024). The combination of propylene glycol and vegetable glycerin e-cigarette aerosols induces airway inflammation and mucus hyperconcentration. Scientific Reports, 14, 1942.
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