TMRE mitochondrial membrane potential assay.

Cells were seeded (5,000) into 96-well plates and incubated for 24 h in normoxia (21% O₂) followed by 24 h in hypoxia (1% O₂). Following 24 h in hypoxia, positive controls received 10 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (dimethyl sulfoxide [DMSO] was used only for DDX28 knockdown [KD] cells and the control) for 20 min to depolarize mitochondria. All cells were then given 200 nM tetramethylrhodamine ethyl ester (TMRE) for 30 min in the dark. Wells were aspirated and washed with 1× PBS-0.2% BSA, and then 100 μl 1× PBS-0.2% BSA was added to each well. Fluorescence was read on a plate reader at an excitation/emission of 544/584 nm.