Figure 7. ERK5 S496 phosphorylation and NRF2 K518 SUMOylation mediate mitochondrial dysfunction in vitro.

(A-F) WT BMDMs and ERK5 S496A KI BMDMs and (G-L) BMDMs transfected with NRF2 K518R and control plasmid, incubated with oxLDL (10 μg/mL) or vehicle. These cells were then seeded on Seahorse plates. After 24 hours, OXPHOS and glycolysis parameters were measured. During extracellular flux analysis, cells were sequentially treated with (A, G) oligomycin (OM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone plus antimycin A (ROT/AA) and used to assess OXPHOS parameters based on oxygen consumption rates. (B, H) The basal respiration, mt ATP production, maximal respiration, and spare respiratory capacity were calculated and plotted as oxygen consumption rates in pmoles/minutes. (C, I) Glucose (GLUC), OM, and 2-deoxyglucose (2-DG) were used to determine glycolysis parameters from extracellular acidification rates. (D, J) Glycolysis, glycolytic reserve, glycolytic capacity, and non-glycolytic acidification were calculated and plotted as the extracellular acidification rate in mpH/minutes. ATP (E, K) and NAD⁺ (F, L) were measured after 24 hrs of oxLDL (10 μg/mL) or vehicle treatment in WT BMDMs and ERK5 S496A KI BMDMs. (M) Proposed model of HC-mediated MC reprogramming to SASP/SAS by ERK5 S496 phosphorylation. The applied statistical tests, sample number, and results in all figures are summarized in Table S3. All data except A, C, G, and I are expressed as mean±SD, and others are expressed as mean±SEM, **P<0.01, *P<0.05.