Fig 2: Effects of resveratrol on mitochondrial biogenesis in kainic acid (KA)-induced status epilepticus in hippocampal CA3 subfield. (A) Pretreated microinjection with resveratrol (Res, 100 µmol) into the hippocampus increased KA-induced NRF1 expression. (B) In accordance with NRF1 protein expression, DNA binding activity of NRF1 measured by electrophoretic mobility shift assay also increased with microinjection with resveratrol (100 µmol) into the hippocampus. (C) Pretreated microinjection with resveratrol (100 µmol) into the hippocampus enhanced Tfam expression 24 h after KA treatment. (D) Mitochondrial protein, COX1, and (E) mtDNA content increased in rats with pretreated microinjection with resveratrol (100 µmol) into the hippocampus. Values are mean ± SEM of quadruplicate analyses from six animals per experimental group. * p < 0.05 versus sham control group, + p < 0.05 versus PBS + KA group in the Scheffé multiple-range test.

Fig 3: Structural consequences of SCAF1 deficiency in the formation of SCs.(A) 2D-BNGE (Dig/DDM) analysis of heart mitochondria resolving complexes and SCs in the first dimension and disrupting SCs into their component complexes in the second dimension. NDUFA9 immunodetection in red indicates the migration of CI, COI immunodetection in red indicates migration of CIV, and CORE2 immunodetection in green indicates migration of CIII. Samples from BL6, CD1, and BL6:S113 heart are compared highlighting the area of migration of the N-respirasome. Red asterisk indicates the absence of the respirasome-derived III2 + IV only in the BL6 sample, indicating that III2 + IV is not physically linked to SCAF1-deficient respirasomes (B) Different traces corresponding to the N-respirasome area of (A). (C) Representation of the two structurally different respirasomes. RS, SCAF1-postive N-respirasome; RnS, SCAF1-negative N-respirasome.

Fig 4: SCs are unstable upon mitochondrial membrane disruption.(A) BNGE resolving complexes and SCs from CD1 liver from intact or digitonized mitochondria preincubated during the indicated time at 4°C and probed with the indicated antibody. (B) BNGE resolving complexes and SCs from CD1 liver from digitonized mitochondria preincubated during the indicated time at 4°C and probed with the indicated antibody. (C) 2D-BNGE/PAGE resolving complexes and SCs in the first dimension and protein components in the second dimension from CD1 liver digitonized preparation preincubated 72 hours at 4°C, showing that SCAF1 is processed (SCAF1*). The membrane was immunoblotted with the indicated antibodies. (D) Structure of SCAF1 sequence, mapping CIII-interacting regions (in green) and CIV-interacting regions (in yellow). The predicted calpain-1 processing site is indicated with a blue line. (E) Quantitative analysis of the SCAF1-derived tryptic (LTSSVTAYDYSGK, in blue) and calpain-1–processed (SSVTAYDYSGK, in orange) peptides. Both peptides were quantified in the BNGE slices corresponding to SCs I + III2 + IV and III2 + IV and to IV2 and to CIV in fresh or in 4°C-incubated liver mitochondria-enriched fractions. The calpain-1–processed peptide was only detected in the nonfresh preparations, attached to CIV and IV2 and also to the respirasome. A structural interpretation of these results is presented at the left; the blue and orange shadows indicate whether the SCAF1 peptide is tryptic or processed, respectively. (F) 2D-BNGE/PAGE showing that the proteolytic cleavage of SCAF1 can be prevented by inhibition of calpain-1. (G) BNGE/PAGE analysis of liver mitochondria showing that the stability of the respirasome and the SC III2 + IV is preserved after digitonization in the presence of a calpain-1 inhibitor. (H) BNGE profile for CIV (COI, red) and SCAF1 (green) in heart samples maintained at 4°C after digitonization during the indicated times.

Fig 5: Protein expressions of COX1 (A) and COX2 (B) in the aortas of wild-type (WT) littermates and eAPP-/- mice. Western blot results are the relative densitometry compared with ß-actin protein (n=10 per group for COX1 and n=8 per group for COX2). All results are representing box plots with whiskers showing the median, 25th to 75th percentiles, and min-max range. * P<0.05 versus young mice of same strain; † P<0.05 versus age-matched WT littermates (two-way ANOVA followed by Tukey’s HSD test).