The LC3‐II flux assay was performed as previously reported with minor modifications (Su et al., 2013 (link)). In brief, mice of the ISO and the CTL groups were subjected to an intraperitoneal injection of 3 μmol/kg BafA1 (Bafilomycin A1, #11038, Cayman Chemical Company) or vehicle CTL (DMSO in saline) 1 h before the ventricular myocardium was sampled for western blot analyses of LC3‐II. The LC3‐II flux is the difference of LC3‐II protein levels between the samples with or without BafA1 treatment of the same group, referring to the net amount of LC3‐II accumulated by BafA1‐induced inhibition of lysosomal function. The LC3‐II flux assays are considered the “gold standard” method for reflecting autophagic activity (Gottlieb et al., 2015 (link)).
Bafilomycin a1
Manufactured by Cayman Chemical
Sourced in United States, Germany
Bafilomycin A1 is a specific and potent inhibitor of vacuolar-type H+-ATPase (V-ATPase). It is a macrolide antibiotic isolated from the culture broth of Streptomyces species.
Automatically generated - may contain errors
Lab products found in correlation
Chloroquine, by Merck Group (7 mentions)
Cycloheximide (chx), by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Bafilomycin b1, by Cayman Chemical (3 mentions)
Wortmannin, by Cayman Chemical (3 mentions)
Mg132, by Cayman Chemical (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Fm4 64, by Thermo Fisher Scientific (3 mentions)
Formic acid, by Thermo Fisher Scientific (3 mentions)
Rapamycin, by Merck Group (2 mentions)
Glutamine, by Merck Group (2 mentions)
Insulin, by Merck Group (2 mentions)
Torin1, by Bio-Techne (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Anti β actin a1978, by Merck Group (2 mentions)
Alexa fluor 594 donkey anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Anti lc3, by Cell Signaling Technology (2 mentions)
74 protocols using bafilomycin a1
1
Measuring Autophagic Flux via LC3-II
2
Antibody Neutralization Assay with Bafilomycin
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Bafilomycin A1, bafilomycin B1 (Cayman Chemical Company), and concanamycin A (Santa Cruz Biotechnology) were resuspended in dimethyl sulfoxide (DMSO) and stored at −20°C until use. Cells were incubated with compounds for 1 h at 37°C prior to infection assay.
Antibody neutralization assays were conducted by preincubating cells with anti-CD164 clone N6B6 (BD Pharmingen) or mouse IgG isotype control (BD Pharmingen) for 1 h at 37°C prior to infection assay. Antibody details can be found inTable S3 .
Antibody neutralization assays were conducted by preincubating cells with anti-CD164 clone N6B6 (BD Pharmingen) or mouse IgG isotype control (BD Pharmingen) for 1 h at 37°C prior to infection assay. Antibody details can be found in
3
Compound Screening in Cell Viability
4
Bafilomycin A1 Inhibition of ZIKV Replication
5
Autophagy Modulation and Pluripotency
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To establish a relationship between autophagy induction and loss of pluripotency, autophagic activity was blocked using autophagy inhibitor Bafilomycin A1 (Cayman Chemical), which inhibits autophagy through both hindering vacuolar acidification required for autophagosome maturation and mTOR activation60 (link),61 (link). Rapamycin (Sigma-Aldrich), an mTOR inhibitor and autophagy inducer62 (link), was utilized for elevating the level of autophagy. Both drugs were used in four different concentrations of 4000, 500, 50, and 5 nM for cytotoxicity analyses. Based on cell viability results, lower concentrations were selected for evaluating viral growth, autophagy, and pluripotency. In influenza-infected conditions, cells were first adsorbed with the virus at MOI of 5.0 for 1 h and then treated with selected concentration of Bafilomycin (5 nM) or Rapamycin (50 nM) mixed in cell-specific culture medium.
6
Cellular Signaling Pathway Modulators
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Forskolin (#1099), 3-isobutyl-1-methylxanthine (IBMX, #2845), and H89 (#2910) were from Tocris. Bafilomycin A1 was from Cayman (#11038). Insulin (#I1507), epinephrine (#E4375), propranolol, (#P0884), isoprenaline (#I6504), dobutamine (#D0676), glucagon (#G2044), and vasopressin (#V0377) were purchased from Sigma. Torin1 was a generous gift from Dr. David Sabatini. Leucine (#L8000) and glutamine (#G3126) were obtained from Sigma.
7
Characterization of CML Cell Lines
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Human CML cell lines, K562 (kind gift from Dr. Junia Melo), KU812 (kind gift from Dr. S Tiong Ong) and murine CML cell lines, 32Dp210 (kind gift from Dr. Brian Druker) and 32Dp210 T315I mutant (kind gift from Dr. James Griffin) were maintained in suspension in RPMI medium (Thermo Fisher Scientific, USA), supplemented with 10% fetal bovine serum, 4 mM L-glutamine (Hyclone, USA), 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific, USA). 32Dp210 and 32Dp210 T315I are murine hematopoietic 32D cells transfected with BCR-ABL1 and T315I mutant respectively [18] (link). The cell lines used in our study are validated with short tandem repeat (STR) profile analysis or Sanger sequencing analysis (Table S1 and Figure S1). Imatinib (LC Laboratories, USA) and ponatinib (Selleckchem, USA) were dissolved in sterile distilled water. Mefloquine hydrochloride (Sigma, US) and bafilomycin A1 (Cayman Chemicals, USA) were reconstituted in dimethylsulfoxide (DMSO; Sigma, USA). N-acetyl cysteine (NAC; Sigma, USA) was dissolved in sterile distilled water. α-Tocopherol (Sigma, USA) was dissolved in a mixture of DMSO and 30% ethanol.
8
Cell Culture and Treatment Protocols
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All cells were cultured at 37°C and 5% CO2. HT1080 (human male), HEK293T/17 (human fetus), RKO (human, gender N/A), Lcells (mouse male), and WNT3A expressing Lcells (mouse male) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and grown in DMEM with 10% FBS. Each cell line was tested for mycoplasma contamination and passaged no more than 20 passages from the original ATCC stock. Cells were treated at the indicated concentrations with the following compounds: CHIR99021 (Cayman Chemicals, Ann Arbor, MI), chlorpromazine HCL (Sigma-Aldrich, St. Louis, MO), bafilomycin A1 (Cayman Chemicals), rhWNT3A (PeproTech, Rocky Hill, NJ), rhWNT5A (R&D, Minneapolis, MN), neomycin trisulfate salt hydrate (Sigma-Aldrich) and carbachol (EMD Chemicals, Gibbstown, NJ).
9
Modulating Autophagy Flux in Cells
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To block lysosomal acidification and subsequent autophagosomal degradation, cells were treated with 200 nM bafilomycin A1 (Cayman Chemical, Ann Arbor, MI) from 1 h p.i. onwards or for the indicated duration of time. To enhance autophagic flux, cells were treated with 300 nM rapamycin (Tocris Bioscience, Bristol, UK) or 5 µM amiodarone hydrochloride (Tocris) from 1 h p.i. onwards. To activate secretory autophagy, cells were pretreated with 500 nM apilimod (AchemBlock, Burlingame, CA) for 16 h, followed by infection in the presence of apilimod for the indicated duration of time.
10
Chemical Optimization for Autophagy Studies
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