Piericidin a

Manufactured by Enzo Life Sciences

Piericidin A is a natural compound isolated from the bacterium Streptomyces piercidinis. It functions as a potent inhibitor of complex I in the mitochondrial electron transport chain.

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Lab products found in correlation

Oligomycin a, by Merck Group (3 mentions) Antimycin a, by Merck Group (2 mentions) Rotenone, by Merck Group (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Oligomycin, by Merck Group (1 mentions) Antimycin, by Merck Group (1 mentions) Seahorse xfe96 analyser, by Agilent Technologies (1 mentions) Piericidin, by Merck Group (1 mentions) Bam15, by Merck Group (1 mentions) Verapamil, by Merck Group (1 mentions) Pmxs ires blasticidin retroviral vector, by Cell Biolabs (1 mentions) Hrp conjugated anti mouse and anti rabbit antibodies, by Santa Cruz Biotechnology (1 mentions) Anti flag m2 magnetic beads m8823, by Merck Group (1 mentions) Blasticidin, by InvivoGen (1 mentions) Human tnf α elisa kit, by Thermo Fisher Scientific (1 mentions) Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions) Mitotracker green fm, by Thermo Fisher Scientific (1 mentions) 2 deoxy d glucose, by Merck Group (1 mentions) 2 nbdg, by Thermo Fisher Scientific (1 mentions)

4 protocols using piericidin a

Seahorse XFe96 Metabolic Assay Protocol

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Intact cell oxygen consumption and extracellular acidification rates (OCR and ECAR) were determined using a Seahorse XFe96 analyser (Agilent). A375 cells were seeded at 1.5 × 10⁴ cells in 100 ml per well in 96-well Seahorse cell culture plates, in DMEM supplemented with 10% FBS (Gibco no. 26140-079) and penicillin/streptomycin (Gibco no. 10378-016). After 14 h, 80 ml of media was removed and 160 ml of HEPES-buffered Seahorse DMEM (Agilent no. 103575-100) supplemented with 10 mM glucose (Sigma no. G8270), 1 mM pyruvate (Agilent 103578-100) and 2 mM glutamine (Agilent 103579-100) was added, and the plate was transferred to a 37 °C non-CO₂ incubator for 1 h. The Seahorse cartridge was hydrated according to the manufacturer’s protocol. oligomycin A (Sigma), BAM15 (Sigma) and piericidin A (Enzo Life Science) + antimycin A (Sigma) were prepared in Seahorse DMEM and added to the cells at final concentrations of 2 mmol l⁻¹ (oligomycin), 2 mmol l⁻¹ (BAM15) and 1 mmol l⁻¹ + 1 mmol l⁻¹ (piericidin + antimycin), respectively. Three baseline respiratory rate measurements were taken, followed by sequential injections of each inhibitor with three measurements each.

To T.L., McCoy J.G., Ostriker N.K., Sandler L.S., Mannella C.A, & Mootha V.K. (2024). PMF-seq: a highly scalable screening strategy for linking genetics to mitochondrial bioenergetics. Nature Metabolism, 6(4), 687-696.

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Retroviral Vector Construction for EGFP-OMP25

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aterials were obtained from the following sources: Anti-FLAG M2 magnetic beads (M8823) from Sigma-Aldrich; anti-HA magnetic beads (88837) from Thermo Fisher Scientific; antibodies to VDAC (4661), CS (14309), RPS6KB1 (2708), GOLGA1 (13192), and CALR (12238) from Cell Signaling Technology; antibody to SHMT2 (HPA-020549) from Sigma-Aldrich; antibodies to LAMP2 (sc-18822) and LMNA (sc-20680) from Santa Cruz Biotechnology; antibody to PEX19 (ab137072) from Abcam; HRP-conjugated anti-mouse and anti-rabbit antibodies from Santa Cruz Biotechnology; piericidin A from Enzo Life Sciences; sodium pyruvate, polybrene, antimycin A, oligomycin A, FCCP, and verapamil from Sigma-Aldrich; blasticidin from Invivogen; and MitoTracker Deep Red FM and TMRM from Thermo Fisher Scientific.
Retroviral 3XFLAG-EGFP-OMP25, 3XMyc-EGFP-OMP25, 3XHA-EGFP-OMP25 vectors were generated via ligation of fragments PCR-amplified from an EGFP-OMP25 construct (Addgene, #38249) (Yoshii et al., 2011 (link)) into a pMXs-IRES-blasticidin retroviral vector (Cell Biolabs) digested with XhoI and NotI.

Chen W.W., Freinkman E., Wang T., Birsoy K, & Sabatini D.M. (2016). Absolute quantification of matrix metabolites reveals the dynamics of mitochondrial metabolism. Cell, 166(5), 1324-1337.e11.

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Cytokine Detection Assay Protocol

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Mdivi-1 (#M0199), Niclosamide (#N3510), 6-Diazo-5-oxo-L-norleucine (#D2141), 2-Deoxy-D-glucose (#D8375), BPTES (#SML0601), Chloroquine (#C6628), 3-Methyladenine (#M9281), Poly-D-lysine hydrobromide (#P7280), Antimycin A (#A8674), Oligomycin A (#O4876) and Rotenone (#R8875) were obtained from Sigma-Aldrich. Olomoucine (#10010240) was obtained from Caymanchem. Piericidin A (#ALX-380-235-M002) was obtained from Enzo Life Sciences. MitoTracker™ Green FM (#M7514), MitoTracker™ Red CMXRos (#M7512) and 2-NBDG (#N13195) were obtained from Thermo Fisher Scientific. EnzyChrom™ Glutamine Assay Kit (#EGLN-100) was purchased from BioAssay Systems. 15-oxospiramilactone (S3) was kindly provided by Prof. Xiaojiang Hao (The State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650204, China). SF2312 was kindly provided by Dr. Florian Muller (The University of Texas MD Anderson Cancer Center, USA).
Cytokine detection–Supernatant was taken from each sample after overnight incubation and analyzed with standard sandwich ELISAs to detect TNF-α using human TNF-α ELISA Kit (#88-7346-22) from Thermo Fisher Scientific and IFN-α (#BMS216INSTCE) from Bender Medsystems, Vienna.

Basit F., Mathan T., Sancho D, & de Vries I.J. (2018). Human Dendritic Cell Subsets Undergo Distinct Metabolic Reprogramming for Immune Response. Frontiers in Immunology, 9, 2489.

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Modulating T-cell Metabolic Pathways

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For certain experiments, IL-2 (10 ng/ml; Miltenyi) was added after 24 h. To bypass initial T cell receptor-dependent signalling, T-cells were activated using PMA (10 ng/ml; Merck) and ionomycin (500 ng/ml; Merck) for 4 h. For partial rescue, T-cells were activated in the presence and absence of canagliflozin, supplemented with dimethyl α-ketoglutarate (0.3 mM; Merck). For complex I inhibition experiments, T-cells were activated in the presence or absence of rotenone (1 μM; Merck) or metformin hydrochloride (10 mM; MedChemExpress). High-dose metformin hydrochloride was used to bypass any transporter-specific uptake. For combined inhibition of complex I and glutamate dehydrogenase, T-cells were activated in the presence and absence of piericidin A (500 nM; Enzo Life Sciences) and R162 (10 μM; Merck). To determine whether ERK inhibition or mTORC1 inhibition phenocopies canagliflozin treatment, T-cells were cultured in the presence of PD-98,059 (25 μM; Merck) and rapamycin (100 nM; Merck), respectively.

Jenkins B.J., Blagih J., Ponce-Garcia F.M., Canavan M., Gudgeon N., Eastham S., Hill D., Hanlon M.M., Ma E.H., Bishop E.L., Rees A., Cronin J.G., Jury E.C., Dimeloe S.K., Veale D.J., Thornton C.A., Vousden K.H., Finlay D.K., Fearon U., Jones G.W., Sinclair L.V., Vincent E.E, & Jones N. (2023). Canagliflozin impairs T cell effector function via metabolic suppression in autoimmunity. Cell metabolism, 35(7).

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