Fig 2: Plk1 self-priming of PBIP1 is influenced by Cyclin A/Cdk1 and MYPT1.(A) Western blots using whole cell lysates from U2OS before (CT) and after si-RNA-mediated Cyclin A-knockdown (CycA KD) of MYPT1 knockdown (MYPT1 KD) for the indicated protein targets. (B) Quantification of Western blots for knockdown efficiency and pPBIP1-Thr78 in the presence or absence of Cyclin A or MYPT1 (p<0.0001, Chi square contingency analysis). (C) U2OS before (Control) and after si-RNA-mediated Cyclin A knockdown (Cyclin A KD) or MYPT1-knockdown (MYPT1 KD) in prometaphase cells stained for DNA, centromere-specific human antisera (ACA), tubulin, and pPBIP1-Thr78 as indicated. Scale bar, 5 µm. (D) Quantification of the intensity of kinetochore pPBIP1-Thr78 staining in U2OS cells before (CT) and after si-RNA-mediated Cyclin A knockdown (CycA KD) or MYPT1-knockdown (MYPT1 KD) in prometaphase cells (n = 100 kinetochores/condition. CT vs CycA KD p=0.01, CT vs MYPT1 KD p=0.001; unpaired, two-tailed t-test).

Fig 3: MYPT1 limits kinetochore Plk1 localization and phosphorylation.(A) U2OS before (CT) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) stained for DNA, centromere-specific human antisera (ACA), tubulin, and Plk1 as indicated. Scale bar, 5 µm. (B) Quantification of the intensity of kinetochore Plk1 staining in U2OS before (Control) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) in prometaphase (Prometa) and metaphase (Meta) cells (n = 100 kinetochores per condition. p<0.0001, unpaired, two-tailed t-test). (C) U2OS before (CT) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) in either prometaphase (Prometa) or metaphase (Meta) stained for DNA, centromere-specific human antisera (ACA), tubulin, and pPlk1-Thr210 as indicated. Scale bar, 5 µm. (D) Quantification of the intensity of kinetochore pPlk1-Thr210 staining in U2OS cells before (Control) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) in prometaphase (Prometa) and metaphase (Meta) cells (n = 100 kinetochores per condition. p<0.0001, MYPT1KD prometaphase vs metaphase, p<0.0001, CT vs MYPT1 KD prometaphase; p=0.01 CT vs MYPT1 KD metaphase; unpaired, two-tailed t-test).

Fig 4: LATS1-mediated phosphorylation of CDC26 modulates the ubiquitination of PLK1 in vivo.(A) The effects of wild-type (WT) and kinase-dead (KD) LATS1 on the ubiquitination of PLK1 in vivo. HEK293T cells were co-transfected with FLAG-tagged PLK1, Myc-tagged ubiquitin and a control vector (mock) or WT or KD LATS1, and then PLK1 was immunoprecipitated using an anti-FLAG antibody. The immunoprecipitates and input fractions were analyzed by immunoblotting with antibodies against c-Myc or FLAG. (B) The effect of a phosphor-mimic mutant of CDC26 (T7D) on the ubiquitination of PLK1 in vivo. HeLa cells were treated with a siRNA targeting the 3’UTR of endogenous CDC26 and then induced to express exogenous wild-type, T7A-mutated, or T7D-mutated CDC26 by adding doxycycline to the culture medium. The cells were then co-transfected with FLAG-tagged PLK1 and Myc-tagged ubiquitin and treated with the proteasome inhibitor MG132 (10 µM) for 12 h prior to cell collection. PLK1 was immunoprecipitated using an anti-FLAG antibody, and the immunoprecipitates and input fractions were analyzed by immunoblotting with antibodies against c-Myc or FLAG.

Fig 5: FancJ binds PLK1 and promotes the activation of PLK1 during the MMC induced centrosome amplification.(A) HA tagged FancJ and GFP tagged PLK1 were co-transfected into 293T cells. An equal amount of cell lysate was used for immunoprecipitation with either IgG or antibody against HA or GFP as indicated. Antibodies used for immunoblotting are indicated on the right. (B) 293T cells were either left untreated or treated with 0.5 µM of MMC for 24 hours. An equal amount of cell lysate was used for immunoprecipitation with either IgG or antibody against PLK1. Antibodies used for immunoblotting are indicated on the right. (C) Either vector or different HA tagged FancJ variants were co-transfected with GFP tagged PLK1 into 293T cells. An equal amount of cell lysate was used for immunoprecipitation with the antibody against GFP. Antibodies used for immunoblotting are indicated on the right. (D) Three different cell lines expressing PLK1 under control of a doxycycline (Dox)-inducible promoter were first transfected with either Control siRNA (C) or pooled siRNA against FancJ (J) and then split into two sets. Doxycycline was added to one set of the cells to induce the expression of different PLK1 variants (+Dox). 24 hours later, cells were collected and used for Western Blot analysis. Antibodies used for immunoblotting are indicated on the right. (E) The three Dox-regulated cell lines were first transfected with either Control siRNA (Control), or pooled siRNA against FancJ (FancJ), and then treated with doxycycline to induce the expression of different PLK1 variants. This was followed by treatment with 0.5 µM MMC for 72 hours. Cells were finally fixed in methanol and stained with antibody against ?-Tubulin. More than 300 cells were counted and the percentage of cells with more than two centrosomes was quantitated. (F) U2-OS cells were first transfected with either Control siRNA or siRNA against FancJ and then split into two sets. One set was treated with 0.5 µM MMC for 72 hours (-BI). In the second set, cells were first treated with 0.5 µM MMC for 12 hours and followed by the addition of 100 nM BI-2536 (+BI). Sixty hours after the addition of BI-2536, cells were finally fixed in methanol and stained with antibody against ?-Tubulin. More than 300 cells were counted and the percentage of cells with more than two centrosomes was quantitated. All error bars are standard deviation obtained from three different experiments. Standard two-sided t test, *P<0.05, **P<0.01, ***P<0.001, NS, no significance.