Fig 2: Cellular levels of Mule are regulated by USP7S. (A) HeLa cells were transfected with USP7S-specific siRNA (200 pmol) for 72 h. (B) HeLa cells were first transfected with 200 pmol USP7S-specific siRNA for 48 h and then transfected with a plasmid encoding siRNA resistant USP7S (0.3 pmol) for further 24 h. (C) HeLa cells were transfected with USP7S (0.6 pmol) expressing plasmid for 24 h. (A–C) Whole-cell extracts were prepared, proteins were separated by 4–20% SDS–PAGE and analysed by western blotting using the indicated antibodies. (D) HeLa cells were transfected with USP7S specific siRNA as previously described, total RNA was prepared and the levels of Mule and GAPDH mRNA were analysed by quantitative real-time polymerase chain reaction in three independent experiments. The knockdown efficiency was confirmed by western blotting. (E) HeLa cells were subjected to the USP7S knockdown as previously described; after 48 h, cells were treated with 2 µM MG-132 for 14 h. Whole-cell extracts were prepared, and proteins were separated by 4–20% SDS–PAGE and analysed by western blotting using the indicated antibodies.

Fig 3: Mule is downregulated after DNA damage, and this is controlled by USP7S. (A–C) HCT116 p53+/+ cells were either left untreated (Contr) or exposed to (A) 150 µM hydrogen peroxide for 15 min, (B) 10 µM etoposide (Eto) for 1 h or (C) 8-Gy ionizing radiation (IR) and harvested at the indicated time points post-treatment. (D) HCT116 p53+/+ cells were transfected with PPM1G siRNA (200 pmol) for 72 h, cells were then harvested either without any additional treatment (−) or 1 h post-treatment with etoposide (+) as previously described. Whole-cell extracts were prepared, proteins were separated by 4–20% SDS–PAGE and analysed by western blotting using the indicated antibodies.

Fig 4: Proposed mechanism for the USP7S-dependent regulation of Mule and Mdm2 protein levels after DNA damage. In response to DNA damage, ATM is activated (1) and stimulates activity of PPM1G (2). PPM1G dephosphorylates and destabilises USP7S (3) that leads to both Mdm2 and Mule self-ubiquitylation and degradation, with the consequent stabilization of p53 and other substrates of Mdm2 and Mule. In unstressed cells, the deubiquitylation enzyme USP7S is stabilized by phosphorylation, and thus stabilizes both Mdm2 and Mule that results in proteasomal degradation of p53.

Fig 5: Mule downregulation after DNA damage is required to coordinate the p53 DNA damage response with DNA repair. (A) HCT116 p53+/+ cells were transfected with Mule-specific siRNA (200 pmol) for 72 h (left panel). Alternatively (right panel), cells were either left untreated (−) or exposed to 8-Gy ionizing radiation (+) and harvested 1 h post-treatment (IR). (B) HCT116 p53+/+ cells were transfected with HA-tagged Mule expressing plasmid (1 pmol) for 24 h and then treated with IR as described in (A). (C) Graphical analysis of the data presented in (B). Statistical data are presented as a mean ± standard deviation, *P < 0.001 as analysed by Student’s t-test from three independent experiments. (D, E) HeLa cells were transfected with HA-tagged Mule HECT-domain expressing plasmid (1 pmol) for 24 h. (E) The cells were then either left untreated (UN) or treated with 25 µM hydrogen peroxide for 5 min, allowed to repair for 0–120 min, and the levels of single strand breaks and alkali labile sites were analysed by the alkaline single-cell gel electrophoresis (Comet) assay. Shown are the mean tail DNA values (%) with standard deviations from three independent experiments. Statistically significant results comparing untreated cells (Control) and Mule plasmid-treated cells (Mule) are represented by *P < 0.01 and **P < 0.001, as analyzed by Student’s t-test. (A, B, D) Whole-cell extracts were prepared, proteins were separated by 4–20% SDS–PAGE and analysed by western blotting using the indicated antibodies.