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. 2019 Oct 10;20(12):e47728. doi: 10.15252/embr.201947728

Figure 5. MITOL is targeted to mitochondria via Tom70 and is then delivered to peroxisomes.

Figure 5

  1. siRNA‐based knockdown of endogenous Tom70, Tom20, Tom40, and Sam50. HeLa cells treated with the corresponding siRNAs and immunoblotted with the indicated antibodies. AIF, apoptosis‐inducing factor.
  2. HeLa cells were immunostained with anti‐Flag and anti‐catalase antibodies after treatment of each siRNA. Tom20‐ and Tom40‐siRNA treatment inhibited import of Su9‐GFP (the targeting sequence of the FoF1 ATPase subunit 9) into the mitochondria, leading to an accumulation of Su9‐GFP precursor proteins in the cytosol and cell nucleus. 3Flag‐MITOL still localized to mitochondria in Tom20, Tom40, and Sam50 siRNA‐treated cells. In contrast, 3Flag‐MITOL dispersed into the cytosol when Tom70 was knocked down. Scale bars, 10 μm.
  3. Statistical analysis of the MITOL subcellular localization following 15 μM CCCP treatment for 3 h in cells treated with control, Tom70, Tom20, Tom40, or Sam50 siRNAs. Su9‐GFP was not imported into the mitochondria, but rather localized to the cytosol and nucleus following Tom20 and Tom40 knockdown. The number of HeLa cells with cytosol‐localized Su9‐GFP or 3Flag‐MITOL in each siRNA experiment was determined. Data represent the mean ± s.e.m. from > 100 cells in three biological replicates. (In case of Tom40, and Sam50 siRNAs, data represent the mean ± s.e.m. from > 60 cells in three biological replicates.) Statistical significance was calculated using a one‐tailed Welch's t‐test.
  4. HeLa cells treated with siControl or siTom70 were fractionated into cytosolic and mitochondrial fractions and then immunoblotted with anti‐lactate dehydrogenase (LDH), anti‐Tom20, and anti‐Flag antibodies. LDH was used as a cytosolic marker. Although some MITOL detached from mitochondria during fractionation, sufficient amounts of MITOL were collected in the mitochondria‐enriched fraction in control cells. In contrast, almost all of the MITOL was collected in the cytosolic fraction in siTom70‐treated cells.