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. 2022 Feb 14;23(4):e53639. doi: 10.15252/embr.202153639

Figure 5. SCAI prevents Polθ‐mediated MMEJ during ICL repair.

Figure 5

  1. pICLpt was replicated in egg extracts immunodepleted with indicated antibodies. Replication samples were amplified by PCR and analyzed by next‐generation sequencing for deletions and insertions. Results from two independent experiments are shown.
  2. Immunoblot analysis of extracts immunodepleted with indicated antibodies. Asterisks denote antibody cross‐reactivity.
  3. pICLpt was replicated in extracts immunodepleted as in (B), digested with PsiI+XhoI or AflIII and resolved on a denaturing polyacrylamide gel.
  4. Immunoblot analysis of Polθ siRNA knockdown efficiency in U2OS cells.
  5. Representative images of metaphase spreads from U2OS/SCAI KO cells transfected with indicated siRNAs and processed as in Fig 1I. Scale bars, 10 µm.
  6. Quantification of radial chromosomes in (E) (mean ± SD; 1,197, 241, 155, and 90 metaphase cells analyzed per condition (left to right) pooled from three independent experiments; *P < 0.05, two‐tailed t‐test).
  7. Quantification of chromosomal breaks/gaps in (E) (mean ± SEM; 150, 150, 114, and 90 metaphase cells analyzed per condition (left to right) pooled from three independent experiments; ****P < 0.0001, Mann–Whitney U test).
  8. Clonogenic survival of U2OS and U2OS/SCAI KO cells transfected with non‐targeting control (CTRL) or Polθ siRNAs and subjected to indicated doses of MMC for 24 h (mean ± SEM; n = 3 independent experiments).