Rotenone

Fourteen-days differentiated Caco-2 cells were used for monolayer permeability experiments, which were all performed in triplicate. Caco-2 cells were incubated with varying concentrations of either vehicle (96% EtOH), CCCP (mitochondrial uncoupler, 0-1-2-3-4-5 μM), rotenone (ROT, Complex I inhibitor, 0-50-100-200 nM), and Piericidin A (PA, Complex I inhibitor, 0-50-100-200 nM; Cayman Chemicals, Ann Arbor, MI, USA) for 27, 30, and 42 h, respectively. Changes in TEER were followed over time to determine permeability of the Caco-2 monolayer. Apical medium was harvested to determine cytotoxicity with the Pierce LDH Cytotoxicity assay kit (Thermo Scientific), according to the manufacturer's instructions.
One hundred μg/mL fluorescein (Sigma Aldrich) was added to the apical medium of Caco-2 cells incubated with vehicle (96% EtOH) or 200 nM PA for 24 h prior to harvesting the basolateral medium at different time points. Caco-2 cells incubated with vehicle (96% EtOH) or 200 nM PA were also harvested at different time points to determine cellular energy status and to isolate RNA for gene expression analysis.

Detection of oxygen consumption rates were analysed in real-time using an XF24 Extracellular Flux Analyser (Agilent Seahorse). Briefly, CSE siRNA transfected HUVEC were plated at 4 × 10⁴ cells/well in V7 24 well plates (Agilent Seahorse) in standard growth media. After attached, cells were treated with AP39 at 100 nM for 24 h. Following, culture media was changed to a non-buffered DMEM media (glucose 10 nM, pyruvate 1 mM and glutamine 2 mM) to allow temperature and pH equilibrium. Oxygen consumption rates (OCR) were measured simultaneously three times to establish baseline measurements. Following, to evaluate mitochondrial function, oligomycin (1 mM) (Sigma Aldrich), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (0.5 mM) (Sigma Aldrich) and a mixture of rotenone and antimycin A (Rot/AntA) (1 mM) (Cayman Chemicals) were injected sequentially, to inhibit the ATP synthase, uncouple oxidative phosphorylation, and estimate non-mitochondrial respiration, respectively. This experiment measures six parameters of the mitochondrial function: basal oxygen consumption, ATP-linked oxygen consumption, proton leak, maximal oxygen consumption, reserve capacity, and non-mitochondrial oxygen consumption as described previously^{23 (link),58 (link)}. After the completion of the determinations, OCR measurements were normalized to protein content by the Bradford method.

Recombinant mouse IL-3, SCF, and IL-33 for in vitro experiments were purchased from Shenandoah Biotechnology (Warwick, PA). Sodium oxamate and 2-deoxyglucose (2DG) were purchased from Alfa Aesar (Tewksbury, MA). Etomoxir, rotenone, and SRT1720 were purchased from Cayman Chemical (Ann Arbor, MI). Antimycin A was purchased from Chem Cruz via Santa Cruz Biotechnology (Dallas, TX). ATP disodium salt was purchased from Tocris via Biotechne Corporation (Minneapolis, MN). Metformin was purchased from MP Biosciences (Santa Ana, CA). A769662 was purchased from Med Chem Express (Monmouth Junction, NJ).

¹³C₅-labeled glutamine (CLM-1822-H), ²H₆-labeled α-ketoglutaric acid (DLM-9476), ¹³C₄-labeled succinic acid (CLM-1571), and ¹⁵N-labeled alanine (NLM-454) were acquired from Cambridge Isotope Laboratories. HPLC-grade reagents, including methanol, chloroform, water, and acetone were acquired from Sigma. Oligomycin (Cayman Chemicals), FCCP (Cayman Chemicals), rotenone (Cayman Chemicals), antimycin A (Sigma), Seahorse XF^e96 XF base media (Agilent), D-glucose (Sigma), sodium chloride (Sigma), L-glutamine (Sigma), sodium pyruvate (Sigma), α-ketoglutaric acid disodium salt (Sigma), dimethyl 2-oxoglutarate (Sigma), DBE-4 (dimethyl-succinate; Sigma), DBE-5 (dimethyl-glutarate; Sigma), 1-octyl-α-ketoglutarate (Cayman Chemicals), methyl pyruvate (Sigma), tert-butyl acetate (Sigma), sodium acetate (Sigma), L-alanine methyl ester hydrochloride (Sigma), L-alanine ethyl ester hydrochloride (Sigma), L-alanine tert-butyl ester hydrochloride (Alfa Aesar), sodium fumarate dibasic (Sigma), dimethyl fumarate (Sigma), diethyl fumarate (Sigma), norvaline (Sigma), deferoxamine mesylate (Cayman Chemicals), doxycycline hyclate (Sigma), hexadimethrine bromide (Sigma), hygromycin B (Sigma), methoxyamine hydrochloride (Sigma), and MTBSTFA + 1% TBDMSCI (Sigma).