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.
Amiodarone hydrochloride
Manufactured by Bio-Techne
Sourced in United Kingdom
Amiodarone hydrochloride is a pharmaceutical compound used in the laboratory setting. It is a class III antiarrhythmic agent with complex pharmacological properties. The core function of amiodarone hydrochloride is to modulate the activity of ion channels in excitable cells, particularly those involved in cardiac electrical activity.
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Lab products found in correlation
Carvedilol, by Merck Group (2 mentions)
Imipramine hydrochloride, by Merck Group (2 mentions)
Thioridazine hydrochloride, by Merck Group (2 mentions)
Rapamycin, by Bio-Techne (1 mentions)
Bafilomycin a1, by Cayman Chemical (1 mentions)
Collagen 1, by Thermo Fisher Scientific (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti p44 42 erk, by Cell Signaling Technology (1 mentions)
Anti cadherin 11, by Thermo Fisher Scientific (1 mentions)
Anti pfak397, by Cell Signaling Technology (1 mentions)
Prolong gold anti fade agent, by Thermo Fisher Scientific (1 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Uric acid, by Merck Group (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Cathepsin b, by Merck Group (1 mentions)
Nigericin, by Bio-Techne (1 mentions)
Ruthenium red, by Bio-Techne (1 mentions)
Llome, by Merck Group (1 mentions)
4 protocols using amiodarone hydrochloride
1
Modulating Autophagy Flux in Cells
2
Cellular Adhesion and Signaling Assay
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Reagents were purchased from Fisher Scientific (Hampton, NH) or Sigma (St. Louis, MO) unless otherwise specified. All reagents were validated by the manufacturer and/or have been previously cited in the literature. Antibodies used were: anti-α-Tubulin (T9026; Sigma, St. Louis, MO), anti-pFAK397 (3283; Cell Signalling Technology, Danvers, MA), anti-pPaxillin118 (44–722 G; ThermoFisher Scientific, Waltham, MA), anti-cadherin-11 (71–7600; ThermoFisher Scientific, Waltham, MA), anti–phospho-p44/42 ERK (Thr202/Tyr204; 4370; Cell Signalling Technology, Danvers, MA), anti-p44/42 ERK (9102; Cell Signalling Technology, Danvers, MA), anti-Ki67 (ab15580; Abcam, Cambridge, UK), AlexaFluor 488 anti-rabbit secondary (A-11,008; ThermoFisher Scientific, Waltham, MA), and AlexaFluor 647 anti-rabbit secondary (A-21,244; ThermoFisher Scientific, Waltham, MA). ECM substrates used were: Collagen I (CB-40,236; Fisher Scientific, Hampton, NH). Inhibitors used were: Amiodarone hydrochloride (40–955–0; Tocris Bioscience, Minneapolis, MN), Carvedilol (C3993, Sigma, St. Louis, MO), Imipramine hydrochloride (I7379; Sigma, St. Louis, MO), and Thioridazine hydrochloride (T9025, Sigma, St. Louis, MO).
3
Cytotoxicity Evaluation of Drug Candidates
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MDA-MB-231 and MDA-MB-468 cells were seeded at 5 × 103 cells/well on uncoated 96 well polystyrene plates allowed to adhere for 24 h. Cells were then treated with various concentrations (0.001, 0.01, 0.1, 1, 5, 10, 50, 100, 500, and 1000 µM) of Amiodarone hydrochloride (40–955–0; Tocris Bioscience, Minneapolis, MN), Carvedilol (C3993,Sigma, St. Louis, MO), Imipramine hydrochloride (I7379; Sigma, St. Louis, MO), and Thioridazine hydrochloride (T9025, Sigma, St. Louis, MO) reconstituted in DMSO according to the manufacturer's instructions, or 1% DMSO and incubated for 3 days. After establishing IC50 values, 5 and 10 µM was chosen for downstream assays.
4
Inflammasome Activation Assay Protocol
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Uric acid, Cytochalasin D, Cathepsin B, LLOMe, KCl and ultrapure LPS were from Sigma. Nigericin, Ruthenium Red and Amiodarone Hydrochloride were purchased from Tocris. Monosodium urate crystals were made as described in Ng et al49 (link). Silica particles were purchased from US Silica Company. Crystals were added to tissue culture grade endotoxin-free water to get the stock solutions for experiments. Mouse Anti-ASC (SC-22514-R), Mouse Anti-Casp-1 (SC-514), Mouse Anti-Cath B (SC-6493) antibodies were purchased from Santa Cruz. Mouse Anti-Casp-1 P20 subunit (AG-20B-0042) was purchased from Adipogen. Anti Casp-1 antibody for Western blotting was from Abcam (EPR4321). HRP-conjugated secondary antibodies and Anti-Rabbit Dylight 549 were purchased from Jackson ImmunoResearch and eBiosciences. Alexa-conjugated secondary antibodies and ProLong Gold antifade agent were purchased from Invitrogen. Mouse Pan-CD45 (17-0451) and LY6G (11-5931) antibodies were from eBioscience. GAPDH antibody (CST 2118) was from Cell Signaling. Mouse Anti IL-1β antibody (AF-401-NA) was from R&D systems. IL-1β and TNFα ELISA ready-set-go kits were from eBioscience Inc. L cell-conditioned media for BMMAC culture was a gift from Robin Yates of the University of Calgary.
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