Suppl. Figure1: RISP and mitochondrial proteins expression in males. (A) Western blots of homogenates from cortex (left column) and hippocampus (right column) of 8-month-old males AD and CIIIKO-AD probing for RISP and Core1 (Complex III), NDUFS3 and NDUFB8 (complex I), SDHA (complex II), Cyt C and COXI (Complex IV) and relative loading controls. (B-C) Quantification of mitochondrial proteins in cortex (B) and hippocampus (C) as a percentage compared to control (indicated as a dashed line). (D-G) Quantification of steady state level of RISP normalized to VDAC1 in cortex (D) and hippocampus (F) and normalized to Core1 in cortex (E) and hippocampus (G) of 8-month-old AD and CIIIKO-AD males. (H) Western blots of homogenates from cortex (left column) and hippocampus (right column) of 8-month-old males AD and CIIIKO-AD probing for mitochondrial membrane proteins VDAC1 and Tim23 and (I) relative quantifications. Loading indicated with $ is repeated, as Rieske, CoreI and VDAC1 were probed on the same gels, therefore share the same loading control.
Bars represent means ±SEM. n=3-4/group, each point is represented by a circle. *p < 0.05; **p < 0.01.
Suppl. Figure2: Complex III deficiency. Spectrophotometric Complex III, Complex IV and Citrate synthase (CS) activity normalized to protein loading, measured in cortical and hippocampal homogenates of 8-month-old AD and CIIIKO-AD females. Bars represent means ±SEM. n=3-4/group, each point is represented by a circle. *p < 0.05; **p < 0.01.
Suppl. Figure3: Neuronal content and Neuroinflammation in males (A) Representative images of H&E staining on histological sections from AD and CIIIKO-AD females and males. (B) Representative images of Nissl staining on histological sections from AD and CIIIKO-AD females and males. (C) Representative images of IHC probing for neuronal marker NeuN staining on coronal sections of 8-month-old CIIIKO-AD and AD males. (D) Western blot probing for Class III β-Tubulin (TUJ1) and Synaptophysin on cortical and hippocampal homogenates from 8-month-old CIIIKO-AD and AD males (n=3-4/group) and relative quantifications. (E) Representative images of IHC probing for glial marker GFAP on cortex (upper row) and hippocampus (lower row) of 8-month-old CIIIKO-AD and AD males. (F) Western blot probing for glial marker GFAP and microglial marker Iba1 on cortical and hippocampal homogenates from 8-month-old CIIIKO-AD and AD males (n=3-4/group) and relative quantifications. *p < 0.05
Suppl. Figure4: proteins involved in degradation pathways. (A) Western blots of homogenates from cortex and hippocampus of 8-month-old females AD and CIIIKO-AD probing for p62 and LC3B I-II and relative quantification (n=3-4/group). (B) Western blots of homogenates from cortex and hippocampus of 8-month-old females AD and CIIIKO-AD probing for 20s proteasome and relative quantification (n=3-4/group).
Suppl. Figure5: Amyloid plaques and Aβ content in males (A) Representative images of coronal sections of AD and CIIIKO-AD males’ brains, immunostained with an anti-Aβ antibody to detect amyloid plaques. (B) Quantification of amyloid plaques in AD and CIIIKO-AD males in cortex and hippocampus (n=3/group). (C) Content of Aβ42 fragments in cortical and hippocampal homogenates from AD and CIIIKO-AD males (n=3-5/group). (D) Western blot analysis detecting the presence of total APP and carboxy-terminal fragment (Ctf) in cortical and hippocampal homogenates from AD and CIIIKO-AD males; quantification of optical density of Ctf/APP signal from Western blot analysis (n=3-4/group).