Fig 2: Mitochondrial remodeling is impaired when autophagic flux is blocked. BAF-treated cells were examined to determine if autophagic flux was required for mitochondrial remodeling. (A) C2C12s were pretreated with BAF or vehicle and then stained for TOMM70A (green) and with Hoechst 33342 (blue) and imaged via fluorescence microscopy. Insets are presented at higher magnification below each original image. Exposure times were individually adjusted to bring out detail. Scale bars: 20 µm. (B) Cells in A were analyzed using Image J to measure mitochondrial pixel area/perimeter ratios of individual cells as a measure of network interconnectivity. (***, P<.001; *****, P<.00001; Student t test, n=10) (C) Western blot analysis of whole cell lysates from BAF-treated C2C12s. (D) Quantification of western blots in C. GM, growth medium.

Fig 3: Loss of PACT augments CL316,243-induced brown adipose mitobiogenesis.A, schematic representation of primary brown adipocyte differentiation protocol (upper panel). Mitochondrial protein levels were analyzed from protein lysates of undifferentiated (Day 0) or differentiated Prkra+/+ or Prkra+/− brown adipocytes by Western blotting using specific antibodies for PGC1α, TFAM, antibody cocktail against ETC proteins, TOM70, NRF1, and β-actin (n = 3). B, the genomic DNA obtained from differentiated brown adipocytes in (A) was analyzed by qRT-PCR for mtDNA (Cox1 or Nd4): nucDNA (ApoB) ratio (n = 3). C, mitochondrial respiration was analyzed by OCR in differentiated Prkra+/+ or Prkra+/− brown adipocytes (n = 6; data were normalized to mitochondrial mass quantified from total OXPHOS protein levels from same samples and represented as arbitrary units (A.U)). D–F, Prkra+/+ or Prkra+/− mice were injected with CL316,243 (1 mg/kg/day) for 6 days and sacrificed 24 h after the final injection (n = 8). D, BAT protein lysates were analyzed by Western blotting using specific antibodies for PGC1α, TFAM, antibody cocktail against ETC proteins, TOM70, NRF1, UCP1, and β-actin. E, mtDNA (Cox1 or Nd4): nucDNA (ApoB) ratio was analyzed by qRT-PCR from total genomic DNA. F, total BAT RNA extracts were analyzed by qRT-PCR for miR-181c and U6 levels. G–I, metabolic parameters of Prkra+/+ or Prkra+/− animals (n = 4): (G) energy expenditure (EE), (H) oxygen consumption (VO2), and (I) respiratory exchange ratio (RER). Protein expression was calculated relative to β-actin (for whole cell lysate) or Ponceau S. (for mitochondrial fraction) and depicted at the top of each blot. Data are mean ± SD. Unpaired t test with Welch’s correction or one-way ANOVA. ∗p ≤ 0.05, ∗∗p ≤ 0.01. BAT, brown adipose tissue; ETC, electron transfer chain; mtDNA, mitochondrial DNA; nucDNA, nuclear DNA; OCR, oxygen consumption rate.

Fig 4: Expression of TOM complex in HT22 cells treated with Aβ for 24 h. Cells were treated with 20μM Aβ25 − 35 for 24 h, with or without followed by treating with 5μM DAPT or 20μM EUK 134 for 24 h. (A) TOM70, TOM40, TOM22, and TOM20 mRNA levels in HT22 cells after 24-h treatment with Aβ25 − 35. (B) TOM70, TOM40, TOM22, and TOM20 protein levels in HT22 cells after 24-h treatment with Aβ25 − 35. (C) Representative bands of western blot of TOM complex in Aβ2535-treated HT22 cells. (D) TOM70 fluorescence of HT22 cells treated with Aβ25 − 35 for 24 h. (E) TOM70 fluorescence relative intensity in HT22 cells after 24-h treatment with Aβ25 − 35. (F) Representative bands of western blot of TOM70 in Aβ25 − 35, DAPT, or EUK 134-treated HT22 cells. (G) TOM70 protein level in Aβ25 − 35, DAPT, or EUK 134-treated HT22 cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Summary of SND1-mediated mitophagy through PGAM5 during tumor progression. (A) Summary: SND1, as an evolutionarily conserved protein, is unexpectedly enriched in the outer and inner membranes of mitochondria through its MTS with the assistance of TOM70. In the mitochondria, SND1 binds to PGAM5 through its C-terminus and this interaction is crucial for SND1-mediated mitophagy, such as the degradation of MFN1, TIM23, and COX4, and dephosphorylation of p-DRP1S637. Finally, SND1-induced advantages of cell proliferation and tumor growth depend on PGAM5-mediated mitophagy and its mitochondrial localization.