The LUAD cell lines A549, HCC827, HCC515, HCC1833, NCI-H1975, NCI-H1993, NCI-H2347 and NCI-H3255 were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences. Cells were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C in an incubator with 5% CO2. A549 cells were grown to ~75% confluence and then transiently transfected with SPAG5 small interfering RNA (siRNA; #1, 5'-GAGGAAAUUGUAGAGCAUTT-3', #2, 5'-CCCGACAACUCACAGAGAATT-3', #3, 5'-CCCGACAACUCACAGAGAATT-3') or scrambled siRNA (5'-UUCUCCGAACGUGUCACGUTT-3') (purchased from Shanghai GenePharma Co., Ltd.) at a concentration of 100 pM using Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Bafilomycin (used at a concentration of 100 nM) was purchased from Cell Signaling Technology, Inc. (cat. no. 54645S).
Bafilomycin
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
Bafilomycin is a specific inhibitor of vacuolar-type H+-ATPase (V-ATPase). It blocks the acidification of cellular organelles, such as lysosomes and endosomes, by inhibiting the proton pump function of V-ATPase.
Automatically generated - may contain errors
Lab products found in correlation
Macs cd14 microbeads, by Miltenyi Biotec (2 mentions)
Recombinant human timp1, by R&D Systems (2 mentions)
Gm6001, by Selleck Chemicals (2 mentions)
Rankl, by Thermo Fisher Scientific (2 mentions)
M csf, by R&D Systems (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Ascorbate, by Merck Group (1 mentions)
Glutamate, by Merck Group (1 mentions)
Erastin, by Merck Group (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Reduced gsh, by Merck Group (1 mentions)
L methionine, by Thermo Fisher Scientific (1 mentions)
L tryptophan, by Thermo Fisher Scientific (1 mentions)
L phenylalanine, by Thermo Fisher Scientific (1 mentions)
L buthionine sulfoximine, by Merck Group (1 mentions)
Sulfasalazine, by Bio-Techne (1 mentions)
Mem amino acids solution, by Thermo Fisher Scientific (1 mentions)
L cystine, by Thermo Fisher Scientific (1 mentions)
5 protocols using bafilomycin
1
SPAG5 Silencing in LUAD Cell Lines
2
Metabolic Modulation during Starvation
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All drugs and supplemented metabolites were added at initiation of starvation, unless otherwise stated. BHAM (150 μM–1.5 mM), oligomycin (10 μM), FCCP (100 nM–5 μM), rotenone (100 nM–1 μM), succinate (10 mM), ascorbate (10 mM), TMPD (100 μM), glutamate (10 mM), malate (10 mM), antimycin A (10 μM), ADP (4 mM), l -cysteine (100 μM–2 mM), cystine, (0.05–1 mM) N-acetyl cysteine (0.5–10 mM), reduced GSH (1–20 mM), 2-deoxyglucose (1–10 mM), koningic acid (10–20 μM), glucose (1–20 mM), NH3 (1–5 mM), NH4Cl (1–10 mM), 3-methyladenine (1–10 mM), MG132 (10 μM), l -buthionine sulfoximine (100 μM–1 mM) and erastin (100 μM) were all from Sigma. Bafilomycin (10–100 nM) was from Cell Signaling. Rapamycin (20–500 nM) was from LC Laboratories. Sulfasalazine (1 mM) was from Tocris Bioscience. MitoParaquat (1–10 μM) was from Abcam. We diluted 10× essential amino acids from 50× MEM amino acids solution (ThermoFisher), for final concentrations of 6 mM l -arginine hydrochloride, 1 mM l -cystine, 2 mM l -histidine hydrochloride-H2O, 4 mM l -isoleucine, 4 mM l -leucine, 4 mM l -lysine hydrochloride, 1 mM l -methionine, 2 mM l -phenylalanine, 4 mM l -tryptophan, 2 mM l -tyrosine and 4 mM l -valine.
3
Measuring Autophagy and Mitophagy Flux
4
Osteoclast Differentiation from Human Monocytes
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Peripheral blood mononuclear cells were isolated from human leucocyte cones (NHS Blood and Transplant, Bristol, UK; anonymous donors) by density gradient centrifugation. CD14 + monocytes were positively selected (MACS CD14 + microbeads; Miltenyi Biotech, Surrey, UK) and seeded at 0.25 × 106 cells/well into 96-well plates containing dentine discs, 1 × 106 cells/well into 48-well plates containing cartilage pieces, or 1 × 106 cells/well into 24-well plates. After overnight adhesion, dentine and cartilage was transferred to new wells. Osteoclast differentiation was induced with 25 ng/ml M-CSF (R&D Systems, Abingdon, UK) and 50 ng/ml RANKL (Peprotech, London, UK) in α-MEM containing 10% FBS, 50 IU/ml penicillin, 50 μg/ml streptomycin sulphate and 2 mM L-glutamine. Differentiation medium was refreshed every 2–3 days for 10–13 days. For inhibitor experiments, mature osteoclasts were treated with bafilomycin (Cell Signalling Technology, Hitchin, UK), E64 (Cambridge Bioscience, Cambridge, UK), GM6001 (Selleckchem, Munich, Germany) or recombinant human TIMP1 (R&D Systems).
5
Osteoclast Differentiation Protocol
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Peripheral blood mononuclear cells were isolated from human leucocyte cones (NHS Blood and Transplant, Bristol, UK) by density gradient centrifugation. CD14+ monocytes were positively selected using MACS CD14+ microbeads (Miltenyi Biotech, Surrey, UK) and seeded at 0.25 x 10 6 cells/well into 96-well plates containing dentine discs, at 1 x 10 6 cells/well into 48-well plates containing cartilage pieces, or at 1 x 10 6 cells/well into 24-well plates. After overnight adhesion, dentine discs and cartilage pieces were transferred to new wells. Osteoclast differentiation was induced by treatment with 25 ng/ml M-CSF (R&D Systems, Abingdon, UK) and 50 ng/ml RANKL (Peprotech, London, UK) in α-MEM containing 10% FBS, 50 IU/ml penicillin, 50 μg/ml streptomycin sulphate and 2 mM L-glutamine. Differentiation medium was refreshed every 2-3 days for 10-13 days.
For inhibitor experiments, mature osteoclasts were treated with bafilomycin (Cell Signalling Technology, Hitchin, UK), E64 (Cambridge Bioscience, Cambridge, UK), GM6001 (Selleckchem, Munich, Germany) or recombinant human TIMP1 (R&D Systems).
For inhibitor experiments, mature osteoclasts were treated with bafilomycin (Cell Signalling Technology, Hitchin, UK), E64 (Cambridge Bioscience, Cambridge, UK), GM6001 (Selleckchem, Munich, Germany) or recombinant human TIMP1 (R&D Systems).
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