All cultures were performed in DMEM with 10% FCS, unless otherwise described. CMT64 cells were obtained from Cell Service at Cancer Research UK. Bovine NPC2 (bNPC2) was added to cultures at 50 μg/ml. Co-cultures of CMT64/IMCs were performed in 12-well plates with 1 × 104 CMT64 cells and 1.5–3 × 105 IMCs. For co-culture of primary AT2 cells with IMC/lung fibroblasts using 6-well Transwell® plates (0.4 μm pore, Corning), 2 × 106 IMCs or 6 × 105 lung fibroblasts were plated into bottom wells, and 3 × 106 AT2 cells were added in transwell inserts. For NPC2 uptake experiments, NPC2-Alexa488 was added to IMCs at 82.5 nM. For bafilomycin A1 (Baf-A1, Sigma), IMCs pre-loaded with bNPC2 (50 μg/ml) for 30 min were chased in serum-free DMEM for 3 h and then incubated with 200 nM Baf-A1 for 24 h.
Bafa1
Manufactured by Merck Group
Sourced in United States, Germany, Sao Tome and Principe
BafA1 is a laboratory equipment product manufactured by Merck Group. It functions as a research tool for studying biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Merck Group (13 mentions)
Chloroquine, by Merck Group (8 mentions)
Rapamycin, by Merck Group (7 mentions)
Bafilomycin a1, by Merck Group (7 mentions)
Nh4cl, by Merck Group (6 mentions)
Cycloheximide (chx), by Merck Group (5 mentions)
Monensin, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Acridine orange, by Merck Group (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Bafilomycin a1 bafa1, by Merck Group (3 mentions)
N acetyl cysteine (nac), by Merck Group (2 mentions)
Z vad fmk, by Selleck Chemicals (2 mentions)
Gapdh, by Abcam (2 mentions)
Dmem medium, by Thermo Fisher Scientific (2 mentions)
Fugene hd, by Promega (2 mentions)
99 protocols using bafa1
1
Interplay of Lung Cells and NPC2 Uptake
2
Cardiac Hypertrophy Protein Turnover Assay
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Bmi‐1 overexpressed AC16 cells or controlled AC16 cells or were planted into 10‐cm plates and treated with Ang II (1 × 10–5 for 70 h) to induce hypertrophy. Cells were cocultured with 5 μM16 MG132 (#474787, Sigma‐Aldrich, USA) or 100 nM28 bafilomycin A1 (Baf‐A1, #S1413, Sigma‐Aldrich, USA) for 12 h, then treated with 100 μM29 cycloheximide (CHX, #5.08739, Sigma‐Aldrich, USA) plus 5 μM MG132, or CHX plus 100 nM Baf‐A1 for indicated times. Then proteins were extracted to detect the target proteins with Western blots.
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Bmi‐1 overexpressed AC16 cells or controlled AC16 cells or were planted into 10‐cm plates and treated with Ang II (1 × 10–5 for 70 h) to induce hypertrophy. Cells were cocultured with 5 μM
3
Zebrafish Oxidative Metabolism Profiling
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An XF24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA) was used to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). 10 of the 24 wells on an islet capture microplate (Agilent Technologies) were used for mutant zebrafish larvae, 10 wells were used for their respective WT controls and 4 wells served as quality control (QC). Four zebrafish larvae were put together in 1 well. Zebrafish larvae were incubated in 1x E3-medium buffered with 4mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Life Technologies, Carlsbad, CA, USA). Seahorse XF Cell Mito Stress kit (Agilent Technologies) was used according to manufacturer’s instructions and modified for zebrafish purposes as described in previous publications [97 (link)–99 (link)]. Baf A1 (1,6 μM final concentration, Sigma-Aldrich) was administered to the zebrafish larvae in order to investigate the effect of Baf A1 on the ORC response. Final DMSO concentration did not exceed 1% in the well of the islet capture microplate. OCR data and ECAR data were exported using Wave Desktop Software (Agilent Technologies).
4
Intestinal Epithelial Cell Response to Lipopolysaccharide
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Human intestinal epithelial Caco-2 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells (passages [22] [23] [24] [25] [26] [27] [28] were maintained in high glucose Dulbecco's modified eagle's medium (DMEM; Gibco, Bethesda, MD, USA) supplemented with 20% fetal bovine serum (FBS; Gibco) under standard conditions (37°C, humidified atmosphere with 5% CO 2 and 95% air). To lower the effects of FBS on cell growth and protection against LPS treatment as well as support cell survival, for LPS treatment, cells were incubated in DMEM containing 0.5% FBS and 0.1 μg/mL LPS (Sigma-Aldrich, St. Louis, MO, USA) for 24 h as described previously [21] . In OCT-treated groups, cells were pre-treated with 10 μM OCT (Selleckchem, Munich, Germany) for 24 h prior to LPS treatments [22] . For stimulation with Bafiomycin A1 (Baf-A1), cells were incubated in DMEM containing 0.5% FBS and 10 nM Baf-A1 (Sigma-Aldrich) for 24 h. For inhibition of TAK1, (5Z)-7-oxozeaenol (0.5 μM, Sigma-Aldrich) was added into culture and the cells were pre-incubated for 6 h.
5
Quantification of Brat Tumor Cells
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L3 brains from UAS-dcr2/UAS-bratRNAi; wor-GAL4, ase-GAL80/+; UAS-stingerRFP were dissected and disrupted (see the Cell dissociation, FACS, sample preparation, and RNA sequencing section). Concentrations of RFP+ brat tumor cells were quantified on a Neubauer cell counter, and ∼1,000 cells were intrathoracically injected into 3–6-d-old adult females with a Nanoject II (Drummond). After a recovery phase of 4 h, flies were injected with either the v-ATPase inhibitor Baf-A1 (9.2 nL of 50 nM Baf-A1; EMD Millipore) or vehicle (DMSO in PBS). Brightfield, GFP, and RFP pictures of living flies were taken on a SteREO Lumar.V12 (Zeiss) with a Pursuit-XS monochrome camera (Diagnostic Instruments) every 24 h after transplantation. GFP autofluorescence signal was subtracted from tumor-specific RFP signal and displayed in false colors black-red-green-blue-cyan-magenta-yellow-white (BRGBCMYW) and subsequently merged with the corresponding brightfield image. The RFP-specific signal was quantified on the outlined area of whole flies after GFP autofluorescence subtraction on ImageJ. The mean intensity of all flies in one picture was plotted.
6
Regulation of Autophagy and Lipid Metabolism
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NAC (1 mM), 3-MA (10 mM), Baf A1 (25 nM), acridine orange (5 μg/ml) and monodansylcadaverine (20 mM) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). LY294002 (10 μM), Triciribine (20 μM), CC (10 μM) and CQ (20 μM) were purchased from Selleckchem (Houston, TX, USA). Antibodies against phospho-mTOR/mTOR, phospho-AKT/AKT, phospho-AMPK/AMPK, LC3, ATG5 and TFEB were purchased from Cell Signaling Technology (Beverly, MA, USA); SQSTM1/p62, ABCA1 and Beclin 1 were from Abcam (Cambridge, UK); ABCG1 and SR-B1 are from NOVUS biologicals (LLC, USA); Lamp2 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); β-actin and horseradish peroxidase-conjugated secondary antibodies were from ZSGB-BIO (Beijing, China). All inhibitors were loaded with cells 1 h before ultrasonic treatment. The inhibitors did not display any cytotoxicity to the cells.
7
Targeting YBX1 in Glioblastoma Cells
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U251 and U87 cells (both 2 × 105) seeded in 35-mm culture dishes were transfected with a final concentration of 50 nM for siRNAs, 500 nM for YBX1-1, and 200 nM for YBX1-2 RNA decoys using Lipofectamine 3000 (Thermo Fisher Scientific). GSCWL1 and GSC456 cells (both 1 × 105) seeded in 60-mm culture dishes were transfected with a final concentration of 200 nM RNA decoys using Lipofectamine RNAiMAX (Thermo Fisher Scientific). U251 cells were treated with CHX (100 μg/mL, Sigma-Aldrich) for 0, 1, 2, 4, 8, or 12 hours, MG132 (100 μM, Sigma-Aldrich) for 8 hours, or Baf A1 (200 nM, Sigma-Aldrich) for 24 hours.
8
Evaluating 5-FU and Autophagy Inhibitors
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5‐FU was obtained from FUJIFILM Wako Pure Chemical. 5‐FU was stored as 100 mM stock solutions in dimethyl sulfoxide (DMSO, Sigma‐Aldrich; Merck KGaA, Darmstadt, Germany) at −20°C. The autophagy inhibitors CQ and BafA1 were bought from Sigma‐Aldrich and LKT Laboratories, respectively. CQ and BafA1 were stored at 60 mM in water and 0.8 mM in DMSO at −20°C, respectively.
9
Cholesterol Accumulation in Macrophages
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Primary BMDMs and iBMDMs were seeded in 24-, 12-, or six-well plates at a density of 0.5 × 106, 1 × 106, or 2 × 106 cells per well, respectively. Experiments with U18666a (662015; EMD Millipore) were optimized in different cell types so as to block lysosomal efflux of cholesterol and achieve absolute lysosomal accumulation. Primary BMDMs exposed to U18666a displayed lysosomal cholesterol accumulation at 48 h, while in iBMDMs, sufficient cholesterol accumulation was observed by 24 h (Fig. S1). Where used, cells were exposed to Torin1 (CAY10997; Cayman Chemical) for 3 h, and Baf A1 (Sigma-Aldrich) and CQ (Sigma-Aldrich) at the indicated concentrations for 1 h before ATP stimulation.
10
Inhibitors of Cell Signaling Pathways
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Baf A1, CQ and LA were purchased from Sigma (Deisenhofen, Germany). The AKT1/2-specific inhibitor AKTVIII was obtained from Merck Millipore (Darmstadt, Germany). The ATM inhibitor KU-55933, the AKT2-specific inhibitor CCT128930 and the pan-caspase inhibitor Z-VAD-FMK were from Selleck Chemicals (Houston, TX, USA).
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