Oxygen consumption rate assay

OCR were measured under a Seahorse instrument (Seahorse Bioscience, Massachusetts, USA) according to the manufacturer’s instructions. Cells were plated in poly-d-lysine-coated XF96 microplates at a density of 5 × 10⁴ cells/well in 100 μL volume of unsupplemented DMEM (no glucose) with or without NCL-EPO-NP. Samples were run with 3 technical replicates per condition. The plates were centrifuged at 1,000 rpm for 4 minutes without use of the centrifuge break. After centrifugation, plate was incubated for 5 minutes before being loaded into a Seahorse XF96 Extracellular Flux Analyzer (Agilent). Basal respiration was measured over 6 hours with 36 ten-minute protocol cycles including a 3-minute mix, 4-minute wait, and 3-minute measurement. After 6 hours, the Complex I and Complex III electron transport chain inhibitors rotenone and antimycin A were injected from Ports A and B at a final concentration of 5 μM to determine nonmitochondrial respiration. Data analysis included subtraction of nonmitochondrial respiration from basal respiration measurements before averaging the last 4 basal measurements.