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. 2023 May 23;63:102752. doi: 10.1016/j.redox.2023.102752

Fig. 7.

Fig. 7

SARS-CoV-2 ORF3a sensitizes cellular ferroptosis through Keap1-NRF2 axis. (A) NCI–H1299 cells were transfected with siKeap1 for 48 h, mRNA level was assayed by qRT-PCR. (B) Tet-on inducible NCI–H1299 cells were transfected with siRNA negative control or siKeap1 for 24 h, then treated with doxycycline for 24 h. Protein level was assayed by immunoblotting. (C) HEK 293T cells were transfected with siRNA negative control or siKeap1 and indicated plasmids for 48 h, and then luciferase activity was determined by Dual Luciferase Reporter Assay. Data show mean ± SEM from three independent experiments. (D) Tet-on inducible NCI–H1299 cells were transfected with siRNA negative control or siKeap1 for 24 h, followed by incubation with doxycycline for 24 h, then cells were treated with erastin, RSL3 and DFO for 8 h, and lipid ROS were determined by flow-cytometry. Data show mean ± SEM from three independent experiments. (E) Tet-on inducible NCI–H1299 cells were transfected with siRNA negative control or siKeap1 for 24 h, followed by treatment with doxycycline for 24 h, then cells were incubated with erastin, RSL3 and DFO for 12 h, and cell viability was determined by CCK8 assay. Data show mean ± SEM from three independent experiments. **P ≤ 0.01, ***P ≤ 0.001, n.s. = not significant.