Figure 6. NRF2 K518 SUMOylation mediated by ERK5 S496 phosphorylation inhibits NRF2 transcriptional activity and subsequent SASP induction in vitro.

(A) BMDMs were transfected with the ERK5 WT, ERK5 S496A, or ERK5b plasmid, and ARE luciferase reporter and the constitutively expressing Renilla luciferase vector. After 16 hours of transfection, cells were pretreated with AX15836 for 1 hour and then treated with ox-LDL (10 μg/ml) or vehicle. After 12 hours, ARE transcriptional activity was measured as described in the Methods. (B) The scheme of NRF2 SUMOylation sites and DNA binding sites. (C) WT BMDMs and ERK5 S496A KI BMDMs were incubated with oxLDL or vehicle. After 0 or 30 min of oxLDL incubation, cell lysates were immunoprecipitated with anti-NRF2 or IgG control and immunoblotted with SUMO2/3 antibody. (D) BMDMs were transfected with GFP-tagged NRF2 K518R mutant or GFP tag plasmid. After 0 or 30 min of oxLDL incubation, cell lysates were immunoprecipitated with anti-NRF2 or IgG control and immunoblotted with SUMO2/3 antibody. The lower graphs represent densitometry data from 5 independent gels, one of which is shown in C and D. (E) WT BMDMs were transfected with NRF K518R mutant or NRF2 WT, the ARE luciferase reporter, and the constitutively expressing Renilla luciferase vector for 16 hours. Cells were treated with oxLDL (10 μg/mL) or vehicle, and 6 hours later, NRF2 transcriptional activity was measured as described in Methods. (F) WT BMDMs were transfected with NRF2 or control siRNA, and after 48 hours of transfection, mtROS levels were detected by MitoNeoD as described in Methods. Cells treated with oxLDL or vehicle were assayed 12 hours later. (G) The percentages of cells positive for SA β-gal staining are shown. More than 200 cells/sample were counted. (H) NF-κB transcriptional activity was measured as described in Fig. 1M in BMDMs transfected with NRF2 K518R mutant or wild type after 24 hrs of oxLDL or vehicle treatment. (I) BMDMs were incubated with oxLDL or vehicle after NRF2 K518R mutant or wild-type transfection. After 24 hours of oxLDL incubation, pHrodo-positive cells were quantified. (J) BMDMs transfected with NRF2 K518R mutant or wild type were treated with oxLDL (10 μg/mL) or vehicle. After 0-24 hours, Western blotting was performed with the indicated antibodies. Representative images from 5 independent experiments are shown. (Quantification was shown in Fig. S8C). The applied statistical tests, sample number, and results in all figures are summarized in Table S3. All data are expressed as mean±SD, **P<0.01, *P<0.05.