Piericidin a

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.

The OCR of lung epithelial cells was measured in a Seahorse XF96 extracellular flux analyser (Agilent Bioscience) with Wave v.2.6.3.5 software. Isolated lung epithelial cells as described above were immediately seeded at 7.5 × 10⁴ cells per well using cell adhesive, Cell-Tak (Corning, catalogue no. 354240) according to the manufacturer’s instructions. Basal mitochondrial respiration was assessed by subtracting the non-mitochondrial OCR, measured with 1 μM antimycin A (Sigma, catalogue no. A8674) and 1 μM piericidin A (Cayman, catalogue no. 15379), from baseline OCR. Coupled respiration was determined by subtracting the OCR in the presence of 2 μΜ oligomycin (Sigma, catalogue no. 75351) from the basal mitochondrial respiration.

NADH:ferricyanide (FeCy), NADH:ubiquinone-1 (Q1), and NADH:decylubiquinone (DQ) activities were measured as described previously (Sazanov et al., 2003 (link)). NADH:FeCy and NADH:Q1 activities of the nanodisc-reconstituted sample were assayed in the buffer containing 10 mM Bis-Tris pH 6.8, 200 mM NaCl, and 10 mM CaCl₂. To increase the solubility of DQ during NADH:DQ assays the assay buffer was supplemented with a small amount of LMNG (0.003%). All the assays for the LMNG-purified sample were performed in the LMNG activity buffer containing 10 mM Bis-Tris pH 6.0, 25 mM NaCl, 10 mM CaCl₂, and 0.1% LMNG.
For the NADH:FeCy assay, 0.9 nM of complex I and 1 mM FeCy (Sigma Aldrich BVBA) was added to the assay buffer in a stirred quartz cuvette. For the NADH:Q1/DQ, 3–9 nM of detergent-solubilized or nanodisc-reconstituted complex I and 100 μM Q1/DQ (Sigma Aldrich BVBA) was added to the assay buffer in a stirred quartz cuvette at 30°C and incubated for 5 min. The reactions were initiated by adding 100 μM NADH (Carl-Roth GmbH) and followed as reduction in absorbance at 340 nm using a Varian Cary 300 UV-Vis spectrophotometer (Agilent Technologies, Inc). To perform the inhibition assay, 20 µM Piericidin A (Cayman Chemical) was added during the NADH:Q1 or NADH:DQ reaction.