Fig. 5.

SLFN11 promotes CDT1 degradation by binding to the CUL4–DDB1 complex in response to replicative DNA damage. (A) Representative interactors of SLFN11. DU145 cells were treated with CPT for 16 h. Nuclear cell lysates were immunoprecipitated with anti-SLFN11 antibody and analyzed by mass spectrometry. (B) HEK293 cells transfected with a DDB1-Flag construct for 2 d were treated with CPT (10 µM) and MG132 (20 µM) for 4 h were lysed and immunoprecipitated with anti-Flag antibody. Interacting proteins were profiled by Western blotting. (C) Interaction between endogenous SLFN11 and DDB1 was assessed by immunoprecipitation with anti-SLFN11 (D2) antibody after treatment with CPT (10 µM) and MG132 (20 µM) for 4 h in DU145 cells. (D, Top) Diagram of DDB1-Flag and SLFN11 deletion mutants. (Middle) 293T cells transfected with DDB1-Flag and indicated SLFN11 constructs were treated with CPT (10 µM) and MG132 (20 µM) for 4 h. Cells were immunoprecipitated with anti-SLFN11 antibody (D2) that detects the N terminus region of SLFN11. (Bottom) Same as above but using anti-SLFN11 antibody (E4) that detects the C terminus region of SLFN11. Interacting proteins were identified by Western blotting. (E) K562 cells stably transfected with vector, SLFN11 and SLFN11-E669Q constructs were treated with CPT (100 nM) and M4344 for 24 h. Proteins were identified by Western blotting. (F) K562 cells stably transfected with vector, SLFN11-WT or SLFN11-E669Q constructs were treated with CPT and M4344 for 72 h. Cell viability was analyzed by CellTiter-Glo. (G) SLFN11-E669Q interacts with DDB1. 293T cells were transiently transfected with DDB1 Flag, SLFN11-WT, and -E669Q for 48 h. Following treatment with CPT (10 µM) and MG132 (20 µM) for 4 h, protein interactions were assessed by IP with anti-SLFN11 (D2) antibody. (H) Diagram of SLFN11 mutations in TCGA. (I) 293T cells transfected with SLFN11-WT, SLFN11-E669Q, or SLFN11-E669K constructs for 48 h, were treated with CPT and M4344 for 72 h. Cell viability was analyzed by CellTiter-Glo.