Fig. 3.
DUB3 promotes the transcriptional activity of NRF2. a DUB3 but not DUB3C89S activates the transcriptional activity of NRF2. HEK293T cells were cotransfected with ARE reporter firefly luciferase (100 ng), pRL-TK (10 ng) and the indicated amounts of DUB3 or DUB3C89S plasmid. Reporter assays were performed 24 h after transfection, and the results are presented as the NRF2/TK luciferase activity. Data were analyzed employing one-way ANOVA and presented as the mean ± SD (N = 3/group). b Reduced DUB3 inhibits NRF2 transcriptional activity. The experiments and data analyses were performed as for panel (a). c Synergistic effects of DUB3 and NRF2 on NRF2 transcriptional activation. The experiments were performed as for panel (a). Data were analyzed employing Student’s t test and presented as the mean ± SD (N = 3/group). d Effects of NRF2 deficiency on the DUB3 induction of NRF2. The experiments were performed as for panel (a). Data were analyzed employing Student’s t test and presented as the mean ± SD (N = 3/group). *P < 0.05 vs. the empty vector group; #P < 0.05 vs. the DUB3 group. e DUB3 overexpression upregulates NRF2 downstream genes. HEK293T cells were transfected with empty vector, DUB3-Flag or DUB3C89S-Flag. mRNA was isolated, and qPCR was performed 48 h after transfection. The mRNA levels of the target genes were normalized to GAPDH, and controls were set equal to 1. Data were analyzed employing one-way ANOVA and presented as the mean ± SD (N = 3/group). f DUB3 deficiency downregulates NRF2 downstream genes. The experiments were performed as for panel (e). Data were analyzed employing one-way ANOVA and presented as the mean ± SD (N = 3/group)