To assess the level of cell death, WT MEFs were incubated with 5 nM Bafilomycin A1 (Millipore-sigma) for 48 h or Rab7−/− MEFs expressing control or Rab27 shRNA lentiviruses were treated with DMSO or 50 nM Bafilomycin A1 or Oligomycin + Antimycin A (10 μM + 10 μM) (Sigma-Aldrich) for 24 h. To assess viability after treatments, cells were incubated with Yo-Pro1 (1:1000; Life Technologies) and Hoechst 33342 (1:1000; Invitrogen) for 15 min at 37 °C before imaging. Percent cell death was determined after treatments by dividing the number of Yo-Pro1-positive cells by total number of Hoechst 33342-positive cells as described58 (link).
Bafilomycin a1
Manufactured by Merck Group
Sourced in United States, Germany, Italy, Sao Tome and Principe, United Kingdom, Macao, Japan, France, Switzerland, Belgium, Australia, China, Israel
Bafilomycin A1 is a macrolide compound that acts as a potent and specific inhibitor of vacuolar-type H+-ATPases (V-ATPases). V-ATPases are involved in the acidification of various intracellular compartments, making Bafilomycin A1 a useful tool for studying cellular processes that rely on pH regulation.
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Lab products found in correlation
Chloroquine, by Merck Group (98 mentions)
Rapamycin, by Merck Group (91 mentions)
Mg132, by Merck Group (70 mentions)
Cycloheximide (chx), by Merck Group (56 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (47 mentions)
3 methyladenine, by Merck Group (42 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (41 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (39 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (34 mentions)
3 methyladenine 3 ma, by Merck Group (31 mentions)
Streptomycin, by Thermo Fisher Scientific (24 mentions)
Penicillin, by Thermo Fisher Scientific (24 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (22 mentions)
Wortmannin, by Merck Group (21 mentions)
Leupeptin, by Merck Group (19 mentions)
Thapsigargin, by Merck Group (18 mentions)
β actin, by Santa Cruz Biotechnology (18 mentions)
Torin1, by Bio-Techne (17 mentions)
Z vad fmk, by Merck Group (16 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (16 mentions)
954 protocols using bafilomycin a1
1
Quantifying cell death in MEFs
2
Measuring V-ATPase Activity via ATPase Assay
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ATPase activity was measured as the liberation of 32Pi from [γ−32P]ATP21 (link). The assay was carried out in a total volume of 200 μl under the following conditions: 4 μl phosphatidylserine (26 μM concentration), 5 μl V-ATPase in buffer C (at 10 nM concentration), and 189 μl ATPase assay solution A (30 mM KCl, 50 mM Tris-MES, pH 7.0, 3 mM MgCl2, and 3 mM [γ−32P]ATP (400 cpm/nmol)) and 2 μl of ethanol or ethanol dissolved bafilomycin A1 (Sigma-Aldrich, 1 μM concentration), The V-ATPase was first incubated with phosphatidylserine for 2 min, and then the reaction was started by addition ATPase assay solution A and ethanol/bafilomycin A1, and continued for 10 min at 37 °C. The ATP hydrolysis reaction was terminated by adding 1.0 ml of 1.25 N perchloric acid, and the released 32Pi was extracted and counted in a Beckman scintillation counter44 (link). The results were expressed as specific activity (μmol of Pi/min per mg of protein), The experiment has been repeated twice, and the results were performed using GraphPad Prism8.
3
Autophagic Pathways in B. abortus Infection
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To study the potential involvement of autophagy pathways in calcium deposition, collagen deposition, MMP-2, RANKL secretion by B. abortus infection in MC3T3-E1 cells, wortmannin or E64 plus leupeptin, bafilomycin A1, or chloroquine (Sigma-Aldrich de Argentina S.A.) were added 2 h before the beginning of infection. wortmannin was used at a concentration of 10 μM, E64 plus leupeptin were used at a concentration of 20 μM, bafilomycin A1 was used at a concentration of 200 nM and chloroquine (Sigma-Aldrich de Argentina S.A.) was used at a concentration of 50 μM (Mizushima et al., 2010 (link)).
4
Autophagy Modulation in Cell Lines
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HKs were treated with 10 μM MG132 (Sigma Aldrich) for 6 hours, 10 nM bafilomycin A1 (Sigma Aldrich) for 8 or 24 hours as indicated in figure legends, in starvation medium (140 mM NaCl, 1 mM MgCl2, 5 mM glucose, and 20 mM Hepes, pH 7.4, modified from [39 (link)]) for 4 hours. HaCaT cells were treated with 10 μM MG132 (Sigma Aldrich), 100 nM bortezomib (Aurogene), or 10 μM epoxomicin (Clinisciences), for 8 hours, starvation medium for 4 hours, 10 μM sorafenib tosylate (Aurogene) for 16 hours in medium without FBS, 10 nM bafilomycin A1 (Sigma Aldrich) for 8 hours, or 10 μM chloroquine (Sigma-Aldrich) for 4 hours. U-2 OS and MCF7 were treated with 10 μM chloroquine for 2 hours. Experimental conditions for all treatments were previously determined in preliminary time-course and dose-response experiments.
5
Epidermal Keratinocyte Isolation and Treatment
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Epidermal keratinocytes in single cell suspensions were prepared from tail and ear of adult mice, by protease digestion. The cells were suspended according to a modification of the method of Hager and colleagues in low-calcium growth medium KGM (Lonza, CC-3111) and plated at about 20% confluence in Costar 12-well plates coated with collagen (Collagen Corp., Vitrogen-100).83 (link) At confluence cells were incubated with MG132 (Sellek, S2619) dissolved in dimethyl sulfoxide (DMSO; Sigma, D8418) and bovine serum albumin (Roth, 00522), conjugated palmitate (Sigma, P9767), and neutralized oleic acid (Sigma, O1008) which was prepared as previously described.15 (link) For inhibition of autophagy, keratinocytes from C57Bl/6 mice were isolated as above and pretreated for 2 h with 100 nM bafilomycin A1 (Sigma, B1793) or its solvent DMSO. Thereafter the medium was exchanged to 50 nM bafilomycin A1 or DMSO in the presence of the other reagents or respective amounts of solvents or carriers and cultivated for 16 h. After incubation cells were harvested for immunoblotting in RIPA buffer containing protease inhibitor cocktail (Sigma, P8340). The induction of cell death was assessed by the quantitative detection of histone-associated DNA fragments in the cell death detection ELISA kit (Roche, 11 544 675 001) of adherent cells. Experiments were performed in triplicate.
6
Murine Macrophage Infection Assay
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Bone marrow cells were flushed from the femurs and tibias of 6–8 weeks old female C57BL/6 mice, and cultured in Petri dishes (2.106 cells/dish) in RPMI 1640 GlutaMax (GIBCO) supplemented with 10% fetal calf serum (FCS, Pan-Biotech) and 20 ng/mL macrophage colony-stimulating factor (M-CSF, Peprotech) at 37°C in the presence of 5% CO2. At day 6, cells were transferred to 24-well plastic plates (2.105 cells/well). For macrophage activation, cells were incubated with 10 ng/mL interferon gamma (IFNγ, Peprotech) and 5 ng/mL LPS (Invivogen) overnight prior to infection. Infection was performed in triplicate at a multiplicity of infection of 0.1 bacterium per cell for 4 h at 37°C. Cells were then washed 2 times with DPBS before addition of fresh medium. At day 0, 2 and 5, cells were lysed in 0.01% Triton X-100 (Sigma), and serial dilutions of the lysates were plated onto 7H11-OADC agar medium for CFU scoring. For infection experiments using Bafilomycin A1 (Sigma), cells were pre-incubated 1 h with 100 nM Bafilomycin A1 prior to infection and removed at 24 h post-infection.
7
Prorenin Signaling Pathway in Renal Cells
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NRK-52E cells (ATCC, Manassas, VA) were cultured and prepared as previously described17 (link). Cells were treated with human recombinant prorenin (Cayman Chemical, Ann Arbor, MI) at 0, 10, 20, 40, 80, and 100 pmol/L for 24 h or 100 pmol/L for 0, 6, 12, 24, and 48 h after preincubation with the Ang II type 1 receptor blocker losartan (10 μmol/L) (Sigma, San Francisco, CA,) or the Ang II type 2 receptor blocker PD123319 (10 μmol/L) (Sigma, San Francisco, CA) for 1 h.
The cells were treated with bafilomycin A1 (1 nmol/L, Sigma-Aldrich), losartan (10 μmol/L), and PD123319 (10 μmol/L) for 1 h before prorenin (100 pmol/L) incubation for 48 h to evaluate the effects of the V-ATPase inhibitor bafilomycin A1 on prorenin-induced FN and α-SMA expression in NRK52E cells. Cells were harvested and used in MTT (3-(4, 5-dimethyl (thiazol-2-yl)-2, 5-diphenyltetrazolium bromide) cell viability, immunoblotting, real-time polymerase chain reaction (PCR), and immunofluorescence assays.
The cells were treated with bafilomycin A1 (1 nmol/L, Sigma-Aldrich), losartan (10 μmol/L), and PD123319 (10 μmol/L) for 1 h before prorenin (100 pmol/L) incubation for 48 h to evaluate the effects of the V-ATPase inhibitor bafilomycin A1 on prorenin-induced FN and α-SMA expression in NRK52E cells. Cells were harvested and used in MTT (3-(4, 5-dimethyl (thiazol-2-yl)-2, 5-diphenyltetrazolium bromide) cell viability, immunoblotting, real-time polymerase chain reaction (PCR), and immunofluorescence assays.
8
Autophagy Modulation in MCF-7 Breast Cancer Cells
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MCF-7 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and used to conduct functional assays. The cells were cultured in DMEM medium supplemented with 10% FBS, 100 μg/mL streptomycin, 100 U/mL penicillin, and 0.03% l -glutamine and maintained at 37 °C with 5% CO2 at a humidified atmosphere. SBI-0206965, 3-MA, bafilomycin A1 and chloroquine were purchased from Sigma–Aldrich [the detail information are as follows: SBI-0206965 (SML1540), 3-MA (189490), bafilomycin A1 (196000) and chloroquine (C6628)]. The primary antibodies used for Western blot are as follows: LC3 B (Abcam, ab192890), beclin-1 (Abcam, ab207612), SF3B3 (Abcam, ab96683), ULK1 (8054, CST, MA, USA), p-ULK1Ser317 (12753, CST), p-ULK1Ser555 (5869, CST), mATG13 (13273, CST), p-mATG13Ser318 (PAB19948, Abnova, Taiwan), ATG101 (13492, CST), FIP200 (12436, CST), SQSTM1/P62 (5114, CST), ATG4B (13507, CST), ATG4C (5262, CST), ATG5 (12994, CST), ATG16L1 (8089, CST), SIRT3 (2627, CST), ac-MnSOD2 K68 (Abcam, ab137037), ac-MnSOD2 K122 (Abcam, ab214675), MMP-2(40994, CST), MMP-9 (13667, CST), E-cadherin (14472, CST), and β-actin (66009-1-Ig, Proteintech, IL, USA). In addition, the secondary infrared antibodies goat anti-rabbit IgG (Cell Signaling Technology #4410, 1:5000) and goat anti mouse IgG (Cell Signaling Technology #4414, 1:5000) were added.
9
Gentamicin Protection Assay for Bacterial Invasion
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T84 cells were seeded on 12- or 24-well plates and infected at a multiplicity of infection (MOI) of 10 bacteria per cell. Gentamicin protection assay was performed as previously described [26 (link)]. Briefly, after 3 h of incubation with the bacteria in the culture medium without antibiotics, the cells were washed with PBS and incubated with the culture medium containing 100 μg/mL of gentamicin for 1 h, 3 h, or 9 h. When indicated, the cells were pre-treated with Bafilomycin A1 (Sigma-Aldrich, Saint-Quentin Fallavier, France) at 50 nM for 30 min before infection, and then Bafilomycin A1 was maintained in the medium during infection. The cells were lysed with 1% Triton X-100 (Sigma-Aldrich, Saint-Quentin Fallavier, France) in deionized water. Samples were serially diluted and plated onto LB agar plates, and the number of bacteria was determined by counting the colony-forming units (CFU).
In the competitive experiment, T84 cells were infected with 10 wild-type LF82 bacteria and 10 mutant bacteria (LF82ΔPAI II or LF82ΔYbtE) per cell as described above.
In the competitive experiment, T84 cells were infected with 10 wild-type LF82 bacteria and 10 mutant bacteria (LF82ΔPAI II or LF82ΔYbtE) per cell as described above.
10
Starvation and Bafilomycin A1 Treatment of HEK293T and HeLa Cells
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HEK293T cells and HeLa cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM; D6546, Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, 173012, Sigma-Aldrich) and 2 mm l -glutamine (25030-081, Gibco, Waltham, MA) at 37°C in a 5% CO2 incubator. For starvation, after being washed twice with phosphate-buffered saline (PBS), cells were cultured in amino acid-free DMEM (048-33575, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) without serum (starvation medium) for the time indicated for each experiment. For bafilomycin A1 treatment, cells were incubated in a starvation medium with 100 nm bafilomycin A1 (B1793, Sigma-Aldrich) for the time indicated for each experiment.
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