Fig. 2.

SARS-CoV-2 ORF3a sensitizes cells to ferroptosis. (A) MEF cells were transfected with indicated plasmids for 24 h and treated with 0.2 μM RSL3 for 8 h, lipid ROS was detected using BODIPY-C11 by flow-cytometry. Data show mean ± SEM from three independent experiments. (B) Tet-on inducible NCI–H1299 cells were induced with different doses of doxycycline (Dox) for 24 h and the protein expression of ORF3a was assessed by Western blot. (C) Tet-on inducible NCI–H1299 cells were treated with erastin (5 μM) or RSL3 (1 μM) for 24 h, and visualized by microscope (Scale Bar =100 μm). (D) Tet-on inducible NCI–H1299 cells were treated with erastin (5 μM) or RSL3 (1 μM) for 24 h, lipid ROS were determined by flow-cytometry and represent image were shown. (E&F) Tet-on inducible NCI–H1299 cells were treated with indicated doses of erastin or RSL3 for 24 h and then cell lipid ROS was detected using BODIPY-C11 by flow-cytometry. Data were analyzed and showed as mean ± SEM from three independently experiments. (G&H) Tet-on inducible NCI–H1299 cells were treated with indicated doses of erastin or RSL3 for 24 h, cell viability was determined by CCK8 assay. Data show mean ± SEM from three independent experiments. (I&J) Tet-on inducible NCI–H1299 cells were treated with erastin (5 μM) or RSL3 (1 μM) in the presence of DFO (100 μM), Fer-1 (1 μM), Z-VAD-FMK (10 μM) and Nec1-s (10 μM) and then cell viability was assessed by CCK8 assay 24 h thereafter. Data show mean ± SEM from three independent experiments. (K) Tet-on inducible NCI–H1299 cells were treated with erastin (5 μM), RSL3 (1 μM) and DFO (100 μM) for 8 h and then lipid ROS level were determined by flow-cytometry. Data show mean ± SEM from three independent experiments. (L) Tet-on inducible NCI–H1299 cells were treated with erastin (5 μM), RSL3 (1 μM) and DFO (100 μM) for 24 h and then cell death was determined with PI staining by flow-cytometry. Data show mean ± SEM from three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***p ≤ 0.001, n.s. = not significant.