Fig. 2.

HACE1 promotes NRF2 activity, stability, and synthesis. (A) Hace1 WT or KO MEFs cotransfected with antioxidative stress response element-luciferase (ARE-LUC) and pRenilla vectors were treated with H₂O₂ (200 μM; 6 h), as indicated. NRF2 activity was determined by measuring luciferase activity. n = 3; *P < 0.01; **P < 0.005. (B) Hace1 WT or KO MEFs were treated with H₂O₂ (200 μM) for the indicated times. Hmox1 and Nqo1 mRNA levels were determined using qRT-PCR. n = 3; *P < 0.01. (C) Hace1 WT or KO MEFs were treated with H₂O₂ (200 μM) for the indicated times. NRF2 protein levels were determined using Western blot. NRF2 levels were normalized to HSC70. n = 3; *P < 0.05. (D) Hace1 WT or KO MEFs were treated with H₂O₂ (200 μM) for the indicated time points. NRF2 localization was determined using immunofluorescence. Typical images are shown on the left. Cells exhibiting nuclear or cytosolic localization were scored. n = 200 from three independent experiments; *P < 0.01. (E) Hace1 WT or KO MEFs transfected with NRF2-expressing or control vector were treated with H₂O₂ (600 μM) or AR (10 μM) for 8 h. Cell death was measured by annexin V and PI staining and presented relative to WT cells. n = 3; *P < 0.01. (F) HEK293T cells were transfected as in E. Cells were pulsed with AHA (250 μM; 1 h) and MG132 to block protein degradation with or without AR (1 μM) as indicated. HACE1 kd in total cell lysates was confirmed using Western blot. Cell lysates were subjected to a Click-it reaction with biotin alkyne followed by streptavidin pulldown, and newly synthetized proteins were detected by Western blot.