Fig 2: Characterization of the MCPH1–CDH1 interaction. A) Schematic diagram of MCPH1 fragments to be used in CDH1 pull-down studies. The sites of potential D-Box and KEN-Box degron sequences are indicated. B) GFP or GFP–MCPH1 variants were transfected into HEK293 cells for 24 h before purification and incubation with FLAG–CDH1 cell extracts (from transfected HEK293 cells). The ability of CDH1 to bind MCPH1-L was investigated by using Western blotting. C) The KEN-Box sequence was mutated to “AAA” in GFP–MCPH1-L and GFP–MCPH1-S before performing the CDH1 pull-down experiment as described in B. D) Schematic diagram of CDH1 fragments to be used in pull-down studies with GFP–MCPH1-L. E) FLAG–CDH1 (or CDH1 fragment) pull-down was performed as in B.

Fig 3: MCPH1 isoforms are phosphorylated during mitosis. A) U2OS cells were treated with 100 ng/ml nocodazole for the indicated time points before Western blot analysis. Asynchronous cells (Asy) were also analyzed. B) U2OS cells were synchronized in prometaphase by treatment with 100 ng/ml nocodazole (20 h). RO3306 (9 µM) was then added to the culture medium for the indicated time points before Western blot analysis. C) U2OS cells were treated with nocodazole (100 ng/ml) for 20 h before Western blot analysis. In the right-hand panel, nocodazole-treated cells were lysed in native lysis buffer before addition (or not) of ?-phosphatase for 20 min at 30°C. Samples were then analyzed by using Western blotting. D) Samples from B were reanalyzed by Western blotting to allow comparison between 2 different MCPH1 antibodies (Cell Signaling Technology/CST vs. Proteintech). Aby, antibody.

Fig 4: Isoform-specific regulation of MCPH1 by APC/C–CDH1. A) HeLa Tet-On cells were cotransfected with GFP–MCPH1-L and either FLAG empty vector or FLAG–CDH1. GFP–MCPH1-L expression was induced by the addition of tetracycline 10 ng/ml. After 24 h, cells were lysed and samples analyzed by using Western blotting. B) HeLa Tet-On cells were cotransfected with GFP–MCPH1-L or GFP–MCPH1-S, along with either FLAG empty vector or FLAG–CDC20 or FLAG–CDH1. GFP–MCPH1 expression was induced and samples processed as in A. C, D) HeLa Tet-ON cells were cotransfected with the indicated plasmids and processed as in A.

Fig 5: MCPH1 isoforms are regulated by APC/C–CDH1. A) A549 cells were treated with 100 ng/ml nocodazole for 20 h before release into fresh culture medium for the indicated time points. ProTAME (12 µM) was also included in one 5 h release sample before Western blot analysis. B) Asynchronous and nocodazole-synchronized cells were treated with 50 µg/ml cycloheximide (CHX) for the indicated time points to block protein synthesis before Western blotting. C) A549 cells were transfected with FLAG empty vector, FLAG–CDH1, or FLAG–CDC20 for 48 h before Western blot analysis. D) A549 cells were transfected with control siRNA or CDH1 siRNA for 72 h before Western blot analysis.