Fig 2: Cdh1 depletion induces apoptosis and G2-M arrest in TNBC cells following PARPi. (A) Flow cytometry analysis showing apoptosis of MDA-MB-468, MDA-MB-231 and T47D cells transduced with scrambled shRNA (NC) or shRNA against Cdh1 in in response to Olaparib (0 µM and 10 µM) for 96 h; (B) the percentage of apoptotic cells were quantified. Data are means of at least three independent experiments. Apoptosis rate was the sum of the two quadrats on the right in each FACS figure. Error bars indicate S.E.M. Significance of differences was calculated using Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: p = 0.05); (C) flow cytometry analysis showing cell cycle distribution of MDA-MB-468 cells transduced with scrambled shRNA (NC) or shRNA against Cdh1 in response to Olaparib (0 µM and 10 µM) for 96 h; (D) quantification of cell cycle distribution. Data are means of at least three independent experiments. Error bars indicate S.E.M. Significance of differences was calculated using Student’s t test (** p < 0.01; ns: p = 0.05); (E) Western blotting analysis the expression of Cdh1, cl-PARP and cl-Caspase 3 in MDA-MB-468 cells transduced with scrambled shRNA (NC) or shRNA against Cdh1 in response to olaparib (0 µM and 10 µM) for 96 h.

Fig 3: Cdh1 depletion sensitizes breast cancer cells to PARPi. (A) Western blotting analysis of Cdh1 in MDA-MB-468, MDA-MB-231, HCC 1806 and T47D cells with Cdh1 depletion via two independent shRNAs; (B) growth analysis of MDA-MB-468, MDA-MB-231, HCC 1806 and T47D cells with or without Cdh1 depletion; (C) Cell viability assay of MDA-MB-468, MDA-MB-231, HCC 1806 and T47D cells transduced with scrambled shRNA (NC) or shRNA against Cdh1 in response to Olaparib (0 µM 0, 5 µM and 10 µM) for 96 h, (** p < 0.01; ns: p =0.05).

Fig 4: USP10 and Cdh1 form a complex with ANLN in a non-competitive manner to balance ANLN levels.A–C The indicated plasmids were transfected into HEK293T cells for 48 h, then cells were harvested with EBC buffer and co-immunoprecipitated using HA or Flag magnetic beads. D The interaction between the recombinant GST-Cdh1 and His-ANLN or His-USP10 was examined using an in vitro GST pull down assay. E–G KYSE150 cells were transfected with siRNAs for 40 h, and then treated with MG132 (20 µM) for 8 h. Immunoprecipitation was used to detect the interaction between endogenous USP10, Cdh1, and ANLN. H HEK293T cells were transfected with the indicated plasmids and treated with MG132 (20 µM) for 8 h before harvesting. Ubiquitination assay was used to detect the ubiquitination level of Flag-ANLN. I KYSE150 cells were transfected with siRNAs for 48 h. The indicated antibodies were used for the western blotting. J KYSE150 cells were treated with DTB and released for different time periods. Interaction between endogenous ANLN and USP10 or Cdh1 was detected by immunoprecipitation. Cyclin B1, E2, and phospho-histone H3 (S10) were used as markers for the different cell-cycle phases. K KYSE150 cells transfected with siRNAs were synchronized and released for different time periods. ANLN levels were detected by western blotting. Cyclin B1 and phospho-histone H3 (S10) were used as markers for mitosis. L–N KYSE150 cells were transfected with siRNAs for 48 h. The indicated antibodies were used for the western blotting. All data are representative of at least three independent experiments.

Fig 5: The APC substrates SKP2 and Cyclin B exhibit a phosphorylation-dependent mechanism for the interaction with core APC during cell cycle progression.a Western blot of HeLa cells synchronized by double thymidine block using phospho-specific antibodies. b Western blot of immunoprecipitates from HeLa cells synchronized by double thymidine block and collected at serial times for immunoprecipitation using SKP2 (upper panel), Cyclin B (middle panel), or ID2 (lower panel) antibodies. E47 and HEB are controls for the immunoprecipitation of endogenous ID2. c Loss of CDH1-mediated regulation of SKP2-S64D stability. Western blot of HeLa cells expressing FLAG-SKP2 WT, S64A, or S64D in the presence or absence of CDH1. d Loss of CDH1-mediated regulation of Cyclin B-S126D stability. Western blot of HeLa cells expressing FLAG-Cyclin B WT, S126A, or S126D in the presence or absence of CDH1. e In vivo ubiquitylation of HeLa cells co-expressing FLAG-SKP2 WT or phospho-mutants and HA-ubiquitin. HA western blot of cellular lyasates immunoprecipitated with FLAG antibody. WCL, whole cellular lysates. f In vivo ubiquitylation of HeLa cells co-expressing FLAG-Cyclin B1 WT or phospho-mutants and HA-ubiquitin. HA western blot of cellular lysates immunoprecipitated with FLAG antibody. WCL whole cellular lysates. g Loss of interaction between the SKP2-S64D and core APC is independent of CDH1. FLAG-immunoprecipitation-western blot of HeLa cells expressing FLAG-SKP2 WT or phospho-mutants in the presence or the absence of CDH1 siRNA (left panel). Right panel, whole cellular lysate (WCL). h Loss of interaction between the Cyclin B-S126D and core APC is independent of CDH1. FLAG-immunoprecipitation-western blot of HeLa cells expressing FLAG-Cyclin B WT or phospho-mutants in the presence or the absence of CDH1 siRNA (left panel). Right panel, whole cellular lysate (WCL). Total and phosphorylated proteins are from independent gels; APC1, APC3, CDH1 are from the same gel in each panel; E47, HEB, pHH3, SKP2 and Cyclin B1 are from independent gels; HA and FLAG/Vinculin in panel e are from different gels; Loading controls are from the same gel as FLAG or HEB in panel a. Molecular weight markers are indicated in kDa. Experiments were repeated two times with similar results.