Fig 2: Plk1 overexpression induces defects in centrosome and chromosome segregation dynamics. a Immunofluorescence against p-Plk1-T210 and pericentrin in MEFs untreated or after 24 h with doxycycline (+Dox). The histogram shows the quantification of mean fluorescence intensity (MFI) of p-Plk1-T210 staining at the centrosomes during late G2 phase (separated; –Dox: n = 54 centrosomes; +Dox: n = 34 centrosomes) or earlier interphase (joint; –Dox: n = 88 centrosomes; +Dox: n = 58 centrosomes; n = 2 replicates). Scale bar, 10 µm. b Immunofluorescence against p-Plk1-T210 and pericentrin in MEFs untreated or after 24 h on Dox. The histogram shows the quantification of mean fluorescence intensity (MFI) of p-Plk1-T210 staining at each mitotic phase [Prometaphase (PM): –Dox n = 22 cells; +Dox: n = 26 cells; Metaphase (M): –Dox n = 17 cells; +Dox: n = 21 cells; Anaphase (A): –Dox n = 15 cells; +Dox: n = 12 cells; Cytokinesis (C): –Dox n = 13 cells; +Dox: n = 15 cells; n = 3 replicates]. c Immunofluorescence against p-Plk1-T210 in primary MEFs untreated or after 24 h on Dox. The histogram shows the quantification of mean fluorescence intensity (MFI) of p-Plk1-T210 staining at individual kinetochores of cells in prometaphase (–Dox: n = 51 kinetochores; +Dox: n = 51 kinetochores; n = 2 replicates) **p < 0.01; Mann–Whitney test. Scale bar, 10 µm. d Immunofluorescence against Sgo1 in primary MEFs untreated or after 24 and 72 h on Dox. The first histogram shows the quantification of mean fluorescence intensity (MFI) of Sgo1 staining in the entire cell at prometaphase (–Dox n = 33 cells; +Dox 24 h: n = 35 cells; +Dox 72 h: n = 52 cells; n = 3 replicates). The second histogram shows the percentage of prometaphase cells with diffused Sgo1 staining (n = 3 replicates). Scale bar, 10 µm. In a, b, d, n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; one-way ANOVA. e Chromosome spreads (DAPI stained) from (+/Plk1);(rtTA/rtTA) MEFs untreated or treated with Dox at the indicated times. Chromosome cohesion was classified in three different status: “parallel arms” as readout of full chromatid cohesion (dark blue box), “separated arms” as readout of chromatid arm separation (mid blue), and “separated sisters” as readout of fully separated chromatids (light blue). (–Dox, n = 24; +Dox 24 h, n = 40; +Dox 48 h, n = 25; +Dox 72 h, n = 22 cells). Scale bars, 20 µm

Fig 3: Differential engagement of PLK1 PBD substrates via the Tyr pocket. (A) FRAP analysis of interphase centrosomes in live cells. GFP-PLK1Wt/AAD/AM at the centrosome was photobleached and its recovery monitored at 0.2 s intervals. The median t1/2 time taken for 50% maximal recovery for GFP-PLK1Wt/AAD/AM were found to be 0.7 s, 0.4 s and 0.3 s respectively. The arrowhead indicates the point of photobleaching. An inset shows data points for the first 5 seconds including photobleaching and recovery time of GFP signal. (B) HeLa cells expressing GFP-PLK1Wt/AAD/AM were synchronized in mitosis by double thymidine block and released as shown in the experimental schedule. The cell lysates were immunoprecipitated using GFP-Trap® beads to pull down GFP-PLK1Wt/AAD/AM and analysed by immunoblotting. The data presented is representative of two independent experiments. (C) HeLa cells expressing GFP-PLK1Wt/AAD/AM were transfected with PBIP1Wt-V5 or pcDNA™3.1/V5-HisA vector (or V5 vector) and harvested 24 h later. PBIP1Wt-V5/ V5 was pulled down from the lysates using V5 antibody and immunoprecipitates were analysed by immunoblotting. Asterisks (*) indicate cross-reacting bands. The data presented is representative of two independent experiments. (D) HeLa GFP-PLK1Wt overexpressing cells were transfected with either PBIP1Wt/F71A/T78A-V5 or V5 vector and harvested 24 h later. PBIP1Wt/F71A/T78A-V5 was pulled down from the lysates using V5 antibody and analysed by immunoblotting. Asterisks (*) indicate cross-reacting bands. The data presented is representative of two independent experiments.

Fig 4: Plk1 overexpression reduces tumor development induced by Kras or Her2 oncogenes. a Percentage of tumor-free mice after doxycycline administration (control, n = 33; Plk1/MMTV-rtTA n = 35; KrasG12D n = 87; KrasG12D/Plk1 n = 40 mice). b Percentage of tumor-free survival after doxycycline administration (control, n = 33; Her2, n = 51; Her2/Plk1, n = 36 mice). In a, b, ****p < 0.0001, Mantel–Cox test. c Number of tumors per animal in the indicated genotypes; **p < 0.01; ****p < 0.0001; Mann–Whitney test. d Interphase-Fluorescent in situ hybridization (I-FISH) on paraffin sections of mammary tumors using two centromeric probes against chromosomes 16 and 17. e Quantification of the frequency of aneuploidy (left) in samples from the indicated genotypes (control, n = 4; Kras, n = 4; Kras/Plk1, n = 3; Her2, n = 4; Her2/Plk1, n = 4 mice). *p < 0.05; ***p < 0.001; one-way ANOVA. f Percentage of cells with 2, 3, or 4 or more chromosomes in the indicated genotypes. g Representative micrographs of Her2 and Her2/Plk1 tumor cells in vitro (H2B-GFP green). Top: mitotic cell with a lagging chromosome. Bottom: cytokinesis failure resulting in binucleation. h Percentage of cells in Her2 tumors (H) and Her2/Plk1 (HP) with the indicated mitotic errors

Fig 5: Overexpression of Plk1 impairs Cep55 and ESCRT loading into the cytokinesis midbody. a (+/Plk1);(rtTA/rtTA) MEFs were untreated (–Dox) or treated for 24 h with doxycycline (+Dox), fixed and stained for a-tubulin (red) and DAPI (DNA, green). Cells in cytokinesis (n > 100 per condition; n = 3 replicates) were evaluated for aberrant cytokinesis, considering the midbody formation and shape, and correct distribution of DNA into the two daughter cells. Scale bars, 10 µm. b The length of the cytokinesis bridge was evaluated by measuring the distance in between the two daughter nuclei in MEFs untreated or after 24 h of Dox treatment (–Dox, n = 17 cells; +Dox, 23 cells). Scale bars, 10 µm. In a, b *, p < 0.05; Student’s t-test. c MEFs expressing a CEP55-EGFP fusion (green) were treated for 24 h with Dox (+Dox), in the absence or presence of 1 µM of the Plk1 inhibitor BI2536 for 1 h at the end of the Dox time (Dox + BI), or left untreated as control (–Dox). a-tubulin is in red. Data represent the percentage of cells with positive CEP55-EGFP signal at the midbody (–Dox, n = 134; +Dox, n = 94; Dox + BI, n = 47 cells; n = 3 replicates (–Dox, +Dox) or 2 replicates (Dox+BI)). Scale bar, 5 µm. d MEFs expressing a Tsg101-mCherry fusion (red) were treated for 24 h with Dox (+Dox), in the absence or presence of 1 µM of BI2536 for 1 h at the end of the Dox time (Dox + BI), or left untreated as control (–Dox). a-tubulin is in green. Data represent Tsg101-mCherry mean intensity at the midbody (–Dox, n = 81; +Dox, n = 60; Dox + BI, n = 37 cells; n = 2 replicates). Scale bars, 5 µm. e MEFs were treated for 24 h with Dox (+Dox), in the absence or presence of 1 µM of BI2536 for 1 h at the end of the Dox time (Dox + BI), or left untreated (–Dox). Cells were stained for a-tubulin (red) and DAPI (DNA, green) and binucleation index was quantified from more than 600 cells in each sample (n = 5 replicates). Scale bars, 50 µm. In c–e, n.s. not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; one-way ANOVA