Figure 2. POR is necessary for PQ-induced cytosolic ROS generation.

(a) Non-targeting (NT) control, POR-null, ATP7A-null, and SLC45A4-null Jurkat cells were treated with 150 μM PQ for 48 hours and intracellular ROS levels were determined as the mean fluorescence intensity (MFI) of CM–DCF. (b) Intracellular ROS levels were measured in empty vector (EV)- or human POR cDNA-reconstituted POR-null Jurkat cells (clone POR_KO2) treated with 50 μM PQ for 48 hours. (c) Extracellular H₂O₂ levels were measured by Amplex red in non-targeting (NT) control, POR-null, ATP7A-null, and SLC45A4-null Jurkat cells following 150 μM PQ for 48 hours. (d) The levels of extracellular H₂O₂ following treatment with 50 μM PQ for 48 hours were measured in the POR_KO2 clone reconstituted with the empty vector (EV) or human POR cDNA. (e) Amplex red was used to measure intracellular H₂O₂ production in saponin-permeabilized non-targeting (NT) control Jurkat cells as well as POR-null, ATP7A-null, and SLC45A4-null Jurkat cells treated with 150 μM PQ for 1 hour. (f) Intracellular H₂O₂ levels were measured in saponin-permeabilized POR-null Jurkat cells (clone POR_KO2) reconstituted with the empty vector (EV) or human POR cDNA following treatment with 150 μM PQ for 1 hour. For all panels, error bars represent SEM (n=4 independent experiments). *, p < 0.05, and **, p < 0.01, compared to control cells. Data were background-corrected to the mean value in untreated cells.