(a) Poly(A)⁺ mRNA, isolated from the tissues of untreated adult mice and from 14-day-old embryos, was analysed by northern blot using ³²P-labelled probes for Plk1 and β-actin, the loading control. Autoradiography was performed at 16 h. The bar graph represents the Plk1 mRNA expression standardized to the β-actin expression (means±s.d., n=5, for each tissue). (b) Total cellular protein prepared from the indicated tissues of untreated adult mice, embryos and NIH3T3 cells were separated by SDS–PAGE and immunoblotted for anti-Plk1, anti-β-actin, α-tubulin and GAPDH as loading controls (means±s.d., n=5, for each tissue). (c) PCR of mouse genomic DNA (tail clip) from wild-type (wt) mice, Plk1-iKD mice (kd) and template DNA as the positive control (pc) are presented. (d) Total cellular protein was prepared from the indicated tissues of Dox-treated adult Plk1-iKD and wt mice and separated by SDS–PAGE for anti-Plk1, -itetR, -Plk3 and -β-actin immunoblotting analyses. (e) Plk1 mRNA levels in the tissues of Dox-treated Plk1-iKD and wt mice relative to the Plk1 mRNA levels in the testis of untreated animals (100%), which was the tissue with the highest Plk1 mRNA expression, are shown (means±s.d., n=5, for each tissue). (f) Relative Plk1 mRNA levels in the tissues of Dox-treated Luc-iKD (Luc-iKD +Dox) animals are compared with the levels in untreated Luc-iKD animals (Luc-iKD -Dox; means±s.d., n=3, for each tissue).

(a–c) Representative examples are shown. (a) Determination of the proliferation index in the mucosal folds of the large intestine: Ki-67-stained cells in the crypts of the transverse mucosal folds of the large intestine of a wt and a Plk1-iKD mouse. Sections are shown at two magnifications. Upper scale bars, 500 μm, lower scale bars, 50 μm. (b) Determination of the proliferation index of the ovarian follicles: Ki-67-stained cells of an ovarian follicle of a wt and of a Plk1-iKD mouse. To calculate the proliferative index in the cyclic ovary, follicles of similar sizes were selected (red boxes). Sections are shown at two magnifications. Upper scale bars, 500 μm, lower scale bars 50 μm. (c) Seminiferous tubules (stage VIII) in the testis of wt and iKD animals (primary magnification: ×20, arrows point to spermatogonia) were Bouin fixed, stained with hematoxylin/eosin or formalin fixed and stained for Ki-67 immunohistochemistry (brown nuclei of spermatogonia) with hematoxylin counterstaining. Scale bar, 50 μm. (d) Ferritin values were determined using an AU400 autoanalyzer (Olympus) in female and male Plk1-iKD mice and a sucrose-treated control group, which were all treated with Dox (means±s.e.m., n=3, for each sample). ***P<0.001, Student's t-test, unpaired and two-tailed.

(Upper) (a) PCR of mouse genomic DNA isolated from MEF derived from wild-type (wt) mice, different Plk1-iKD (kd) clones (kd12, 17, 18) and a template DNA as the positive control (pc) are shown. (Middle) Western blot analysis of Plk1 and Plk3 expression in different MEF clones (wt, kd) at increasing Dox concentrations is shown. (Lower) Western blot analysis of Plk1 expression and proliferation of different MEF clones (wt, kd) at increasing Dox concentrations (0–80 μg ml⁻¹) is shown. For standardization, β-actin expression was chosen. The uptake of fluorescein-labelled Plk1 siRNA, Plk1 mRNA depletion and proliferative activity of primary human cells (b) HUVEC, (c) fibroblasts and (d) keratinocytes are shown. (Right) FACS analysis of control cells (that is, incubated with Gene Silencer and non-labelled siRNA MM) and fluorescein-labelled siRNA-transfected cells. The concentration of the Plk1 siRNA is indicated at the top of each panel. Transfections were conducted for 6 h. The intensity of the fluorescence, which represents the uptake of fluorescein-labelled Plk1 siRNA, is indicated in the lower right corner. (Left) The western blot analysis of the Plk1 and the Plk3 protein was conducted 24 h after transfection of the primary human cells with Plk1 siRNA and MM siRNA at the indicated concentrations. β-Actin served as a loading control. Camptothecin (Camp) and/or nocodazole (Noc) controls were included. Plk1 protein expression in primary cells (HUVEC, fibroblasts, keratinocytes) was standardized to the expression of β-actin and is relative to the level of Plk1 in untreated cells (100%). (Right) Proliferative activity of primary cells at different siRNA concentrations (0–100 nM; means±s.d., n=3, for each time point). *P<0.05, Student's t-test, unpaired and two-tailed.

Primary cells (a) HUVEC and (b) fibroblasts and (c) keratinocytes were transfected with siRNA MM or siRNA Plk1 at different concentrations (50–100 nM) and analysed by FACS at 24 and 48 h. Representative histograms and the quantification of the cell-cycle analysis are shown. (d) Keratinocytes were labelled using Plk1, pericentrin and α-tubulin antibodies and 4,6-diamidino-2-phenylindole (DAPI) for the analysis by immunofluorescence microscopy. Representative examples of siRNA MM- and siRNA Plk1-transfected keratinocytes are depicted. The mitotic index and the percentage of cells with aberrant mitotic spindles were determined by counting 250–300 cells (means±s.d., n=5, for each sample of cells). Scale bar, 5 μm. (e) Plk1-depleted keratinocytes were labelled using BubR1 and α-tubulin antibodies and DAPI for the analysis by immunofluorescence microscopy. Representative examples of Plk1-transfected keratinocytes in different mitotic stages are depicted. Scale bar, 5 μm.

(a) (Upper) Correlation of the Dox (0–1,000 ng ml⁻¹)-induced knockdown of the Plk1 protein and the induction of apoptosis in lysates of wt and transgenic ES cells treated with Dox for 46 or 70 h are shown. The immunoblot for Plk1, caspase-3, PARP and β-actin is also presented. (Lower) Caspase-3 activity was determined in the cell lysates using the Caspase-Glo 3/7 Assay (means±s.d., n=3, for each concentration). (Upper) Lysates of Plk1 siRNA-transfected (b) HUVEC, (c) fibroblasts and (d) keratinocytes were immunoblotted for Plk1, PARP and β-actin. Camptothecin (Camp) and/or nocodazole (Noc) controls are included. (Middle) Caspase-3 activity was determined in the cell lysates using the Caspase-Glo 3/7 Assay (means±s.d., n=3, for each concentration). (Lower) Annexin V-based measurements of cells treated with siRNA Plk1 or a siRNA control (siRNA MM) were correlated with levels of apoptosis. (Upper) Lysates of siRNA (Plk1 and/or p53)-transfected (e) keratinocytes and (f) HUVEC cells were immunoblotted for Plk1, p53, p-p53(S15) and β-actin. Camptothecin (Camp) controls are included. (Lower) Caspase-3 activity was determined in the cell lysates using the Caspase-Glo 3/7 Assay (means±s.d., n=3, for each concentration).

(a) Proliferative activity of MEFs at different BI 2536 concentrations (25–250 nM; means±s.d., n=3, for each concentration). *P<0.05, **P<0.01, ***P<0.001, Student's t-test, unpaired and two-tailed. (b) FACS profile of exponentially growing MEFs treated for 24 h with BI 2536 (10 nM–2 μM). Percentage of cells in G₂/M are given at each concentration. (c) Kinase activity of immunoprecipitated Plk1 protein from MEFs treated for 24 h with BI 2536 (10 nM–2 μM) using the cytoplasmic retention signal (CRS) of cyclin B1 as substrate. (d) MEFs were labelled using Plk1, pericentrin and α-tubulin antibodies and 4,6-diamidino-2-phenylindole for the analysis by immunofluorescence microscopy. Representative examples of BI 2536-treated and control MEFs are depicted. Scale bar, 5 μm. The mitotic index and the percentage of cells with aberrant mitotic spindles were determined by counting 250–300 cells (means±s.d., n=5, for each sample of cells). (e) The correlation of BI 2536-mediated inhibition of the Plk1 and the induction of apoptosis in MEFs was analysed. The immunoblot for Plk1, β-actin and PARP is presented. (f) Annexin V-based measurements and caspase activity were determined in cell lysates of BI 2536-treated MEFs (24 h) using the Caspase-Glo 3/7 Assay (means±s.d., n=3, for each concentration).

(a) Plk1 mRNA and protein levels in Plk1-iKD- and Luc-iKD-ES cells (±Dox) determined by semi-quantitative RT-PCR analysis and western blot. (b) Volumes of teratocarcinomas derived from Plk1-iKD- and Luc-iKD-ES cells (inoculated on opposite flanks of the same animal) either after induction with Dox or in the absence of Dox starting with 3 days before inoculation. Significant inhibition of tumour growth (P<0.05) is indicated with an asterisk (means±s.d., n=12, for each time point). (c) Representative image of a mouse exhibiting Plk1-iKD- and Luc-iKD-ES cell-derived tumour growth on opposite flanks. (d) Total cellular protein was prepared from Plk1-iKD- and Luc-iKD-ES cell tumours and separated by SDS–PAGE for anti-Plk1, -itetR and -β-actin immunoblotting analyses (means±s.d., n=3, for each treatment). *P<0.05, Student's t-test, unpaired and two-tailed.