Rotenone

Manufactured by Thermo Fisher Scientific

Sourced in United States

Rotenone is a laboratory chemical commonly used as a pesticide and biocide. It is a naturally occurring compound extracted from the roots of certain tropical plants. Rotenone is primarily used as a research tool to study mitochondrial function and cell death.

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Lab products found in correlation

16 protocols using rotenone

Mitochondrial Poisons in Cell Assays

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Two mitochondrial poisons were used, 6-OHDA (cat. H4381 Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and rotenone (Cat. MKBS1062V, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). rotenone was dissolved in dimethyl sulfoxide (DMSO, Thermo Fisher Scientific, Hampton, NH, USA) while 6-OHDA is water-soluble. The final concentration of DMSO used was never higher than 0.1% (v/v). Cell treatment strategies are specified in the respective results subsection and in the respective figure legend.

Simões R.F., Oliveira P.J., Cunha-Oliveira T, & Pereira F.B. (2022). Evaluation of 6-Hydroxydopamine and Rotenone In Vitro Neurotoxicity on Differentiated SH-SY5Y Cells Using Applied Computational Statistics. International Journal of Molecular Sciences, 23(6), 3009.

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Caco-2 Cell Bioenergetics Profiling

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The Seahorse XF-96 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA) was used for determination of real-time oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using materials purchased from Agilent Technologies. 2.5x10⁴ Caco-2 cells were cultured for 24 hours in XF-96 Seahorse plates (Agilent Technologies Santa Clara, CA, USA), washed with PBS and the media changed to DMEM containing 100 μg/ml penicillin, 100 μg/ml streptomycin, phenol red and 25 mM glucose + 1mM pyruvate + 2mM glutamine (complete medium) or 5mM Sodium butyrate (butyrate medium) (Thermo Fisher Scientific, Walthman, CA USA). Three measurements were performed over 15 minutes in the basal condition, upon addition of 1μM oligomycin, 1μM FCCP, and 1μM rotenone + 1μM antimycin A (Thermo Fisher Scientific, Walthman, CA USA). The basal respiratory rate (BRR), maximal respiratory rate (MRR) and spare respiratory capacity (SRC) were calculated according to the manufacturer’s instructions.

Martín-Adrados B., Wculek S.K., Fernández-Bravo S., Torres-Ruiz R., Valle-Noguera A., Gomez-Sánchez M.J., Hernández-Walias J.C., Ferreira F.M., Corraliza A.M., Sancho D., Esteban V., Rodriguez-Perales S., Cruz-Adalia A., Nakaya H.I., Salas A., Bernardo D., Campos-Martín Y., Martínez-Zamorano E., Muñoz-López D., Gómez del Moral M., Cubero F.J., Blumberg R.S, & Martínez-Naves E. (2023). Expression of HMGCS2 in intestinal epithelial cells is downregulated in inflammatory bowel disease associated with endoplasmic reticulum stress. Frontiers in Immunology, 14, 1185517.

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Cell Line Sourcing and Validation

Cited 2 times

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Cell lines were sourced and cultured as described previously (12 (link),18 (link),26 (link)). A673 (ATCC Cat# CRL-1598, RRID: CVCL_0080) and SK-N-MC (ATCC Cat# CRL-2270, RRID: CVCL_1398) cell lines were obtained from ATCC, TC-32 from Dr. Timothy Triche (Children’s Hospital of Los Angeles), and SK-N-MC from Dr. Shu-Fang Jia (University of Texas, Houston, TX). Cell lines were validated yearly using STR profiling. Cell lines were tested for the presence of Mycoplasma using the Universal Mycoplasma testing kit every 3–4 months as needed and were most recently tested in December 2021. Cells that were freshly thawed were used up to two months in culture. Monoclonal cell lines generated in this study were expanded and stored long-term in 90% Fetal Bovine Serum (FBS) and 10% DMSO in liquid nitrogen. Soft agar assays were performed as described (12 (link),18 (link),26 (link)). SP-2509 was purchased from Cayman Chemicals. Antimycin A, rotenone, and oligomycin were purchased from Thermo-Fisher. All oligonucleotides used in this study were purchased from Integrate DNA Technologies (IDT), or Thermo-Fisher Scientific.

Tokarsky E.J., Crow J.C., Guenther L.M., Sherman J., Taslim C., Alexe G., Pishas K.I., Rask G., Justis B.S., Kasumova A., Stegmaier K., Lessnick S.L, & Theisen E.R. (2022). Mitochondrial Dysfunction Is a Driver of SP-2509 Drug Resistance in Ewing Sarcoma. Molecular Cancer Research, 20(7), 1035-1046.

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Metabolic Modulation in Immune Cells

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All reagents were from Sigma unless otherwise stated. Specific kits and linked reagents are described below. Non-canonical reagents are listed.
Rotenone, dehydroepiandrosterone (DHEA), thenoyltrifluoroacetone (TTFA), antimycin A, 2-deoxyglucose (2-DG), oligomycin, glutamate (pH 7.4 with NaOH), zymosan particles from Saccharomyces cerevisiae (including pHrodo, Thermo-Fisher), Yeast from Saccharomyces cerevisiae, LPS (lipopolysaccharide), luminol, DTAC (Dodecyltrimethylammonium chloride), Accutase, diazo-oxo-norleucine (DON), bis-phenylacetamido-thiadiazolyl-ethyl sulphide (BPTES), atpenin A5 (Cayman chemicals), dimethylmalonate, UK-5099, etomoxir, phorbol–myristate–acetate (PMA), mitotracker red CMX-Ros (Thermo-Fisher), all-trans retinoic acid, M-CSF (Peprotech), VAS-2870, Sotrastaurin (Cayman chemicals), N-Formylmethionyl-leucyl-phenylalanine (fMLF), Pam3CSK4 (Invivogen), uric acid crystals, R848 (Invivogen), 4′,6-Diamidino-2-Phenylindole (DAPI, Thermo-Fisher), Hoescht 33342 (Thermo-Fisher), 2-mercaptoethanol.

Davies L.C., Rice C.M., Palmieri E.M., Taylor P.R., Kuhns D.B, & McVicar D.W. (2017). Peritoneal tissue-resident macrophages are metabolically poised to engage microbes using tissue-niche fuels. Nature Communications, 8, 2074.

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Mitochondrial Respiration Assay Protocol

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Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, C2920), oligomycin (OM, O4867, antimycin A (AA, A8674), rotenone (Rot, R8875), monensin sodium salt (MON, M5273), 2-deoxyglucose (2-DG, D6134), Concanavalin A (Con A, C2010), bovine serum albumin (BSA, A6003), sodium chloride (S9888) and Roswell Park Memorial Institute (RPMI) 1640 medium without phenol red and HEPES (11835030), Dulbecco’s phosphate-buffered saline (DPBS, 14190094), and Hanks’ Balanced Salt Solution (HBSS, 14175095) were purchased from Thermo Fisher Scientific (Pittsburgh, PA). Extracellular flux (XF) RPMI assay medium pH 7.4 (103576-100), XF 1.0 M glucose (103576-100), XF 100 mM pyruvate (103578-100), and XF 200 mM glutamine (103579-100) were purchased from Seahorse Biosciences, Agilent Technologies (Santa Clara, CA).

Janssen J.J., Lagerwaard B., Porbahaie M., Nieuwenhuizen A.G., Savelkoul H.F., van Neerven R.J., Keijer J, & de Boer V.C. (2022). Extracellular flux analyses reveal differences in mitochondrial PBMC metabolism between high-fit and low-fit females. American Journal of Physiology - Endocrinology and Metabolism, 322(2), E141-E153.

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Cell Line Validation and Soft Agar Assay

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Cell lines were sourced and cultured as described previously (12, 18, 26 (link)). A673 (ATCC Cat# CRL-1598, RRID: CVCL_0080) and SK-N-MC (ATCC Cat# CRL-2270, RRID: CVCL_1398) cell lines were obtained from the ATCC, TC-32 from Dr. Timothy Triche (Children's Hospital of Los Angeles, Los Angeles, CA), and SK-N-MC from Dr. Shu-Fang Jia (University of Texas, Houston, TX). Cell lines were validated yearly using STR profiling. Cell lines were tested for the presence of Mycoplasma using the Universal Mycoplasma testing kit every 3 to 4 months as needed and were most recently tested in December 2021. Cells that were freshly thawed were used up to two months in culture. Monoclonal cell lines generated in this study were expanded and stored long-term in 90% FBS and 10% DMSO in liquid nitrogen. Soft agar assays were performed as described previously (12, 18, 26 (link)). SP-2509 was purchased from Cayman Chemicals. Antimycin A, rotenone, and oligomycin were purchased from Thermo Fisher Scientific. All oligonucleotides used in this study were purchased from Integrate DNA Technologies, or Thermo-Fisher Scientific.

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Mitochondrial Dysfunction Evaluation Protocols

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After overnight cell plating, cells were challenged with dimethyl sulfoxide (DMSO; D2650; Sigma) as vehicle control or 1 µM Rotenone (R8875; Sigma-Aldrich) or 10 µM Antimycin A (A8674; Sigma-Aldrich) for the indicated time points. For treatment in transient knockdown or overexpressing conditions, cells were transfected with siRNA/plasmid DNA using RNAi Max reagent/LipofectamineTM 2000 (13778150/11668019; Thermo Fisher Scientific) or transduced with adenovirus for 48 h. Then, the cells were challenged with DMSO or 1 µM Rotenone or 10 µM Antimycin A for the indicated time points. For hypoxic condition, HK2 cells were plated overnight and transfected with siRNA for 48 h. Then, the cells were exposed to hypoxic condition (1% O₂) for 1 h using INVIVO₂ 400 (Hypoxia workstation).

Thoudam T., Chanda D., Sinam I.S., Kim B.G., Kim M.J., Oh C.J., Lee J.Y., Kim M.J., Park S.Y., Lee S.Y., Jung M.K., Mun J.Y., Harris R.A., Ishihara N., Jeon J.H, & Lee I.K. (2022). Noncanonical PDK4 action alters mitochondrial dynamics to affect the cellular respiratory status. Proceedings of the National Academy of Sciences of the United States of America, 119(34), e2120157119.

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Mitochondrial Dysfunction and Cell Death Analysis

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CM-H₂-DCFDA, JC-1, rotenone, antimycin-A and both reduced and non-reduced Mito Tracker were obtained from Invitrogen (Thermo Fisher Scientific, Inc.). The antibodies against PHB1 and cyclooxygenase (COX) IV were purchased from Abcam, while the Annexin V-FITC apoptosis kit was obtained from Becton, Dickinson and Company. The antibodies against β-actin, cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase-3 were obtained from Cell Signaling Technology, Inc. and DAPI from MilliporeSigma.

Zhang L, & He Y. (2022). Prohibitin inhibits high glucose-induced apoptosis via maintaining mitochondrial function in human retinal capillary endothelial cells. Experimental and Therapeutic Medicine, 23(6), 427.

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Adenovirus-mediated α-Synuclein Overexpression

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Adenovirus overexpressing human wild-type SNCA was kindly gifted from the lab of S.J.L. COS7 cells were infected with the adenovirus overexpressing human α-syn at 0.8 × 10^8 pfu and incubated at 37 °C for 24 h. When the confluency reached 90%, the cells were split onto 6-well plates for further experiments. The α-syn aggregation was induced by the treatment of 100 nM rotenone (Sigma-Aldrich, R8875) for 56 h. For the MitoTracker analyses in SH-SY5Y α-syn A53T cells, rotenone was treated at 1 µM for 48 h with the co-treatment of 100 nM ATC161 for 24 h, then MitoTracker Red CMXRos (Invitrogen; M7512) was used to stain intact mitochondria following the manufacturer’s instruction.

Lee J., Sung K.W., Bae E.J., Yoon D., Kim D., Lee J.S., Park D.H., Park D.Y., Mun S.R., Kwon S.C., Kim H.Y., Min J.O., Lee S.J., Suh Y.H, & Kwon Y.T. (2023). Targeted degradation of ⍺-synuclein aggregates in Parkinson’s disease using the AUTOTAC technology. Molecular Neurodegeneration, 18, 41.

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Mitochondrial ROS Measurement in Neutrophils

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For measurement of mitochondrial ROS (mtROS) release, 1 × 10⁶ neutrophils were incubated with 10 μM rotenone (Tocris Bioscience, Bristol, UK), 200 μm MitoTEMPO (Cayman Chemical, Ann Arbor, MI, USA), 10 μM TX or TX plus rotenone and loaded with 2.5 μM MitoSOX™ Red Mitochondrial Superoxide Indicator (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 30 min. After incubation, MitoSOX™ fluorescence intensity was measured using a BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) at wavelengths of 510 nm excitation and 580 nm emission.

Salinas C., Barriga K., Albornoz A., Alarcon P., Quiroga J., Uberti B., Sarmiento J., Henriquez C., Ehrenfeld P., Burgos R.A, & Moran G. (2023). Tamoxifen triggers the in vitro release of neutrophil extracellular traps in healthy horses. Frontiers in Veterinary Science, 9, 1025249.

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