Skip to main content
. 2018 Feb 9;19(3):e42139. doi: 10.15252/embr.201642139

Figure 4. Recomplemented human TDP2 localizes to the mitochondria of DT40 cells and protects against the mitochondria‐specific TOP2 poison, mtDox.

Figure 4

  1. Representative immunoblots of mitochondrial (Lanes 2–4) and nuclear extracts (Lanes 5–7) from wild‐type (WT), TDP2 knockout (TDP2 −/−/−), and human TDP2‐complemented (hTDP2) DT40 cells (15 μg per lane). Recombinant human TDP2 bearing a His6‐tag was loaded as a marker (Lane 1). Upper panel probes for TDP2 protein (#: non‐specific bands), middle panel for nuclear TOP1 (nuclear maker), and bottom panel for COX4 (mitochondrial marker). The 70‐kDa TOP1 band represents a commonly observed proteolytic truncation product.
  2. Proteinase K protection assay of purified mitochondria from hTDP2 DT40 cells. Upper panel probes for TDP2 (#: non‐specific band). Lower panel probes for MRPL12, an internal mitochondrial marker.
  3. TDP2 activity in mitochondrial (Mito) and nuclear fractions. Upper panel: schematic representation of the assay: TDP2 cleaves the 5′‐phosphotyrosine bond (5′‐Y) and generates a product bearing a 5′‐phosphate group (5′‐P). Lower panel: the same samples as in (A) were tested for TDP2 activity. Indicated extracts were serially diluted (1:3 serial dilution starting at 0.5 μg/μl) and incubated with 32P‐3′‐end‐labeled duplex DNA constructs for 2 hours. Left lanes: markers bearing a 5′‐phosphate or a 5′‐hydroxyl group.
  4. Clonogenic survival assays of wild‐type (WT), TDP2 knockout (TDP2 −/−/−), and human TDP2‐recomplemented (hTDP2) DT40 cells treated with indicated concentrations of mtDox for 14 days (n = 2).
  5. Protective effect of TDP2 on mtDNA. The indicated DT40 cell lines were treated with mtDox for 8 h followed by genomic DNA extraction. mtDNA copy numbers were measured by quantitative real‐time PCR normalized to a nuclear housekeeping gene (18S‐rRNA). Relative mtDNA copy numbers were normalized to mock‐treated control cells (n = 7, error bars represent SEM). *P ≤ 0.05 (two‐tailed Wilcoxon test).

Source data are available online for this figure.