Fig 2: High expression of MTCH2 in glioma cells may increase mitochondrial OXPHOs. a, b Western blots and quantifications showing the enriched expression of MTCH2 in glioma cells compared to primary neurons and astrocytes. c qPCR results showing the decreased mRNA level of MTCH2 in A172 cells by siRNA-mediated knockdown (N = 3). d, e Western blots and quantifications showing the decreased protein level of MTCH2 in A172 cells by siRNA-mediated knockdown. Noted that other mitochondrial outer membrane proteins Tom20 and Tom40 were not affected by MTCH2 knockdown. f Immunostaining images showing the mitochondrial localization of MTCH2 in A172 cells. Noted that MTCH2 signals were dramatically decreased by siRNA-mediated knockdown. Scale bar, 10 µm. g Western blots showing the increased mitochondrial respiratory proteins and 4-HNE levels by MTCH2 knockdown in A172 cells

Fig 3: Mitochondrial Membrane Potential (ΔΨm) is disrupted in Aged Macrophages during S. pneumoniae Infection. Young and aged macrophages were cultured with S. pneumoniae (MOI = 25). (A) Cell viability was quantified at 4 and 24 hours post infection using the MT cell viability assay. Light production by metabolically active cells was proportional to the number of live cells in culture (t-test: ***P < 0.001 and ****P < 0.0001). (B) At 60 and 120 minutes post S. pneumoniae infection, cells were collected and changes in mitochondrial membrane potential (ΔΨm) were assessed by flow cytometric changes in MitoCapture red fluorescence. (C) % of MitoCapture red positive cells and (D) mean fluorescence intensity (MFI) was assessed for each sample (t-test: **P < 0.01 and ****P < 0.0001). (E) DNA was isolated from uninfected and S. pneumoniae infected macrophages at 24 hours post infection and the ratio of mtDNA to nuclear DNA levels were assessed by real time PCR (t-test, **P < 0.01). (F) ATP concentration in young and aged macrophages was assessed at baseline and in response to 4 hours of infection (t-test: ****P < 0.0001 and one-way ANOVA ****P < 0.0001). (G) mRNA was isolated from young and aged macrophages at select time points of S. pneumoniae infection and gene expression was analyzed by real time PCR using RT2 Profiler Assays (Mouse Mitochondrial Energy Metabolism PAMM-008ZA). Specific mRNA expression of genes associated with complex I (NDUF, NADH:Ubiquinone Oxioreductase), complex II (SDH, succinate dehydrogenase), complex III (UQCR, ubiquinol-cytochrome C reductase), complex IV (COX, cytochrome C oxidase), and complex V (ATP synthase) were quantified (t-test: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 and one-way ANOVA ****P < 0.0001). (H) Mitochondria were isolated from young and aged macrophages at select time points post infection and protein expression was assessed using the Total OXPHOS antibody cocktail. The blot was stripped and re-probed for TOM20 expression to confirm equivalent protein input between lanes. Full-length gels and densitometric analysis using Image J software are included in Supplemental Fig. 1 for comparison. Similar results were obtained from at least three independent experiments. Data are expressed as the mean ± SD.

Fig 4: MITOL specifically relocates to peroxisomes Schematic topology representation of the mitochondrial membrane proteins analyzed in this study.Peroxisomal translocation of other mitochondrial proteins such as MitoNEET/CISD1, Fis1, Miro1/2, and Tom70 (mitochondrial outer membrane proteins), and Hsp60 (mitochondrial matrix protein) was not observed in CCCP-treated cells. HeLa cells stably expressing HA-Parkin were treated with 15 µM CCCP for 3 h, and then immunostained with anti-MitoNEET/CISD1, anti-Fis1, anti-Miro1/2, anti-Tom70, anti-Hsp60, and anti-catalase antibodies. Scale bars, 10 µm.A multi-spanning outer membrane protein PBR (peripheral benzodiazepine receptor) did not co-localize with catalase, but did with Tom20 during mitophagy. HeLa cells stably expressing HA-Parkin were treated with 15 µM CCCP for 3 h, and then immunostained with anti-Flag, anti-Tom20, and anti-catalase antibodies. Scale bars, 10 µm.

Fig 5: Human CLU_45 kDa is translated from an AUG start site in Exon 3.(A) Empty vector (pCMV6), V5-tagged pCMV6-human CLU Exon 2–9, or Exon 3–9 constructs were transfected into human SH-SY5Y cells. Cell lysates were harvested and probed for CLU immunoreactivity using anti-V5 (left panel) or anti-Clusterin (right panel). (B: left panel) pCMV6-human CLU Exon 2–9 or Exon 3–9 constructs were transfected into human SH-SY5Y cells. The resulting cell lysates were treated with PNGase F or Endo H and probed for CLU immunoreactivity using anti-V5. (B: right panel) Cell lysates harvested from pCMV6-human CLU Exon 3–9-transfected SH-SY5Y cells were treated with O-glycosidase, neuraminidase, or a combination of the two and probed for expression of CLU_45 kDa protein using anti-V5. (C) pCMV6-human CLU Exon 3–9-transfected SH-SY5Y cells were subjected to cellular fractionation using the Qiagen Qproteome Mitochondrial Isolation Kit and analyzed for CLU immunoreactivity using anti-V5. Fraction isolation was confirmed using calnexin (ER), Gapdh (cytosol), lamin A/C and lamin B1 (nucleus), and VDAC and Tom20 (mitochondria).