Coxiv

Proteins were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membranes, and blocked in 5% skimmed milk for 1 h. The membranes were then incubated overnight at 4°C with the following primary antibodies (anti-rabbit or anti-mouse): cleaved caspase3 (1 : 1,000; Affinity, China), Tom20 (1 : 1,000; BD, USA), COXIV (1 : 1,000; Affinity, China), LC3B (1 : 500; Affinity, China), P62 (1 : 1,000; Affinity, China), Bcl-2 (1 : 1,000; Affinity, China), Bad (1 : 1,000; Affinity, China), Parkin (1 : 1,000; Proteintech, USA), PINK1 (1 : 1,000; Proteintech, USA), Drp1 (1 : 1,000; Affinity, China), Mfn2 (1 : 1,000; Affinity, China), Atg-5 (1 : 800; Affinity, China), β-actin (1 : 5,000; Affinity, China), GAPDH (1 : 5,000; Affinity, China), Bcl-xl (1 : 1,000; Affinity, China), Bcl-w (1 : 1,000; Affinity, China), Bim (1 : 1,000; Affinity, China), procaspase3 (1 : 1000; Abcam, USA), and Tubulin (1 : 5,000; Affinity, China). Membranes were then incubated for 2 h with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary detection antibodies (1 : 5,000). Immunoreactivity was detected by using an enhanced chemiluminescence detection system (Beyotime, Haimen, China) and visualized on an imaging system (Kodak, Shanghai, China).

FGF21 was purchased from PeproTech. The miR‐130 agomir was obtained from RiboBio. The primary antibodies used in this study included PPARγ (Abcam, #ab178860), α‐SMA (Abcam, #ab5694), bcl‐2 (Abcam, #ab59348), proliferating cell nuclear antigen (PCNA; Cell Signalling Technology, #13110), β‐actin (Cell Signalling Technology, #4970), Bax (Cell Signalling Technology, #14796), caspase‐3 (Cell Signalling Technology, #9665), cleaved caspase‐3 (Cell Signalling Technology, #9665), apoptosis‐inducing factor (AIF; Cell Signalling Technology, #5318), cytochrome c (Cyt C; Cell Signalling Technology, #11940), cyclin‐dependent kinase 1 (CDK1; Affinity Biosciences, DF6024), cyclin D1 (Affinity Biosciences, AF0931) and COX IV (Affinity Biosciences, AF5468). The PPARγ agonist pioglitazone (Pio) was obtained from Selleck (Houston, TX, USA). Donkey anti‐mouse IgG H&L (Alexa Fluor 488; lot no. ab150105) antibodies were purchased from Abcam. Horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG antibody (lot no. BL003A) was purchased from Biosharp. Tissue Mitochondria Isolation Kit (#C3606) and Cell Mitochondria Isolation Kit (#C3601) were purchased from Beyotime. The One‐Step Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) Apoptosis Assay Kit (#C1089) was purchased from Beyotime.

Total tissue protein, mitochondrial protein, and nuclear protein were extracted from hippocampal tissue, quantified, separated using denaturing polyacrylamide gel electrophoresis (SDS‐PAGE), and transferred onto a PVDF membrane. The membranes were incubated overnight at 4°C with the following primary antibodies: SV2A (1:1000; Abcam, USA), PAR (1:1500; Santa Cruz Biotechnology, USA), AIF (1:2000; Abcam, USA), PARP1 (1:1500; Proteintech, Wuhan, China), H2AX (1:1000; Santa Cruz Biotechnology, USA), γH2AX (1:1500; Santa Cruz Biotechnology, USA), COX‐IV (1:2500; Affinity, Jiangsu, China), Histone (1:3000; Proteintech, Wuhan, China), and β‐actin (1:7000; Proteintech, Wuhan, China). The membranes were washed with TBST and then incubated with a horseradish peroxidase‐conjugated secondary antibody for 60 min. An enhanced chemiluminescence kit was used to visualize the blots. Band density was analyzed using Image J software, and the relative band density of the target protein was normalized to β‐actin, COX IV, or Histone.