Fig. 4.

IR inhibited both OXPHOS and glycolysis via the p90RSK and ERK5 S496 phosphorylation.

(A-D, F–I) BMDMs isolated from WT mice were pre-treated with FMK-MEA (10 μM) or vehicle for 1 h and exposed to IR (A-D). Alternatively, BMDMs from WT and ERK5 S496A KI mice were exposed to IR (F–I). These cells were then seeded on Seahorse plates. After 24 h, OXPHOS and glycolysis parameters were measured. During extracellular flux analysis, cells were sequentially treated with (A, F) Oligomycin (OM), carbonyl cyanide 4-(tri 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, mitochondrial ATP production, maximal respiration, and spare respiratory capacity were calculated and plotted as oxygen consumption rates in pmoles/minutes. Mean ± SD, (n = 3). (C, G) Glucose (GLUC), OM, and 2-deoxyglucose (2-DG) were used to determine glycolysis parameters from extracellular acidification rates. (D, I) Glycolysis, glycolytic reserve, glycolytic capacity, and non-glycolytic acidification were calculated and plotted as the extracellular acidification rate in mpH/minutes. Mean ± SD, (n = 3). (E, J) BMDMs from WT mice were pre-treated with FMK-MEA (10 μM) or vehicle for 1 h and then exposed to IR (E); alternatively, BMDMs from WT and ERK5 S496A KI mice were exposed to IR (J). **P < 0.01 (K, L) BMDMs from WT mice were pre-treated with FMK-MEA (10 μM) or vehicle for 1 h and then exposed to IR, and after 24 h of IR, the expressions of M1- (K) and M2 (L) -like markers mRNA levels were detected by the fold inductions relative to the expression in unexposed BMDMs. Mean ± SD, (n = 3).