Isolation of total RNA from murine tissues
After Dox-treatment wt and Plk1-iKD mice were rapidly killed. Organs were prepared and stored in RNAlater Stabilization Reagent (Qiagen). For the RNA preparation, tissue pieces of 20 mg were dispersed with a PCR Tissue Homogenizing Kit (Omni International). Then total RNA was isolated using an RNeasy Mini Kit (Qiagen). The RNA was stored at concentrations of 50 ng μl−1 to 1 μg μl−1 at −20 °C.
Plk1 expression analysis
For northern blot experiments, 2 μg poly(A)+ RNA was subjected to 1.5% denaturating agarose gel-electrophoresis. The RNA was blotted to a Hybond-N+ nitrocellulose membrane (Amersham Biosciences) overnight by capillary forces. Subsequently, the membranes were heated at 80 °C (2 h) to fix the RNA. The mRNA (Plk1, β-actin) was hybridized using a radioactive probe-containing α-[P32]-dCTP (1×106 c.p.m. ml−1). Probes for detecting the Plk1 mRNA were generated by standard PCR protocols using the sense primer 5′-TAATGACTCAACACGCCTGATT-3′ and the antisense primer 5′-AGCTCAGCAGCTTGTCTACCAT-3′. The β-actin-specific probe was generated using the sense primer 5′-GAGGAGCACCCCGTGCTGCTGACCGAGGCC-3′ and the antisense primer 5′-CCTGCTTGCTGATCCACATCTGCTGGAAGGTG-3′.
To quantify Plk1 transcripts, 1 μg of RNA was subjected to qRT-PCR (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems). The qRT-PCR was conducted using a TaqMan Gene Expression Assay for murine Plk1 (Applied Biosystems). As an internal control in the TaqMan Assay, murine β-actin was used (Applied Biosystems).
This assay was performed using a standard TaqMan PCR Kit protocol in Applied Biosystems Step One. The 10 μl PCR reaction included 0.67 μl RT product, 1× TaqMan Universal PCR Master Mix, 0.2 μM TaqMan probe, 1.5 μM forward primer and 0.7 μM reverse primer. The reactions were incubated in a 384-well plate at 95 °C (10 min), followed by 40 cycles of 95 °C (15 s) and 60 °C (1 min). All reactions were run in triplicate.
Preparation of primary cells and cell culture
MEFs were prepared from 15-day-old embryos by a standard procedure51
. Splenocytes and bone marrow cells were isolated by standard protocols51
. For Dox-treatment ES cells were cultured in DMEM (Invitrogen) containing 10% FCS from PAA, 4,500 mg l−1
glucose, 1× non-essential amino acids and various concentrations of Dox (Sigma). The Dox-medium was prepared and changed each day.
For the isolation of HUVEC, an umbilical vein was cannulated with a sterile needle and perfused by PBS to remove remaining blood for subsequent incubation with α-chymotrypsin (Sigma). The umbilical cord was then flushed with RPMI 1640 medium (Invitrogen). After centrifugation, cells were resuspended and cultured in RPMI 1640 medium with 75 μg ml−1 endothelial cell growth supplement (Sigma). Fibroblasts were cultured in DMEM with 10% FCS and keratinocytes in Dermalife K Medium Complete Kit (Cell Systems) at 37 °C and 5% CO2 in a humidified environment. Research studies involving the use of human specimens were reviewed and approved by the Institutional Review Board of the Goethe-University School of Medicine.
Cancer cell lines were purchased from the American Type Culture Collection and grown in the recommended medium supplemented with 10% FCS at 37 °C and 5% CO2 in a humidified environment. SW480 (colon), MDA-MB-231 (breast) and 293T (kidney/embryonic +T) cells were grown in DMEM, HeLa (cervix) cells in MEM and SK-OV-3 (ovarian) cells in McCoy's 5a. Media were obtained from Invitrogen and McCoy's was from Biochrom.
One day before transfection, 2×105
Art4.12 ES cells carrying the FRT/F3 configuration at rosa26
were plated on a 3-cm dish in 2 ml medium20
. Before transfection 2 ml fresh medium was added. A volume of 3 μl FuGENE 6 Reagent (Roche) were mixed with 100 μl serum-free medium (OptiMEM 1 with Glutamax-I, Invitrogen) and incubated (5 min). A volume of 100 μl of the FuGENE 6/OptiMEM solution was added to 2-μg circular DNA (c
=0.33 μg μl−1
) and incubated (15 min). Fresh medium was added to the transfected cells the following day. From day 2 on, the medium was changed daily, replaced by medium containing 250 μg ml−1
G418 (Geneticin, Invitrogen). The siRNA against Plk1 and the control siRNA MM31
were purchased from Sigma.
Normal cells were transfected with siRNA (10–150 nM) using Gene Silencer (BioCat). Cells were collected after 24 or 48 h. The transfection efficiency was ascertained by transfecting FITC-labelled siRNA (6 h) followed by FACS analysis (FACScan, BD Biosciences). Cancer cells were transfected with siRNAs (0.5–10 nM) using the Oligofectamine protocol (Invitrogen). Control cells were treated as indicated for each experiment. Cells were collected 24 h after transfection for western blot, cell-cycle analysis and the apoptosis assay. For cell-cycle kinetics, the HeLa and SW480 cells were collected 0, 3, 6, 9, 12, 15 and 18 h after the addition of siRNAs. Proliferation was measured 0, 24, 48, 72 and 96 h after siRNA transfection.
Cells were incubated with 2 mM thymidine (Sigma; 16 h), washed extensively with PBS and released in fresh medium (8 h), followed by a second thymidine block (16 h)12
Investigating the cell-cycle distribution by FACS analysis
Cells were seeded, transfected and collected after 24 and 48 h or every 3 h for the cell-cycle kinetics. Cells were trypsinized, washed with PBS and fixed in 70% ice-cold ethanol (30 min). After centrifugation, the cells were resuspended in PBS and incubated with 300 μg ml−1 RNAse A and 30 μg ml−1 propidium iodide (30 min) at 37 °C. Finally, the cell-cycle quantification was performed by flow cytometry (FACScan, BD Biosciences) using ModFit (Verity Software House).
Cells were grown on a glass coverslip, fixed and permeabilized with methanol (−20 °C), and washed with PBS before the addition of appropriate primary and secondary antibodies. Microscopy was performed using a Zeiss Axio Imager.Z1 microscope (Zeiss) equipped with a ×40 objective, and the images were captured and processed using AxioVision Software (Zeiss).
Analysis of apoptosis
The Vybrant Apoptosis Assay Kit no. 2 (Alexa Fluor 488 annexin V/propidium iodide staining, Invitrogen) was used, and the cells were analysed by flow cytometry using a FACScan (BD Biosciences)53
. The activity of caspase-3/7 was determined using the Caspase-Glo 3/7 Assay according to the manufacturer's instructions (Promega).
The following antibodies were used: mouse monoclonal antibodies against Plk1 (1:1,000), Cdk1 (1:1,000), cyclin B1 (1:5,000), p53 (1:2,000; Santa Cruz), Plk2 (1:500), β-actin (1:200,000; Sigma), Plk3 (1:1,000; BD Pharmingen), α-tubulin (1:500,000; Cedarlane/Biozol), Emi1 (1:500; Invitrogen, Karlsruhe), Securin (1:1,000; Abcam) and Ki-67 (1:100; DAKO), and rabbit polyclonal antibodies against PARP (1:1,000), Plk4 (1:1,000; Cell Signaling Technology/New England Biolabs), GAPDH (1:1,000; Abcam), Plk1 (1:1,000), pericentrin (1:500), caspase-3 (1:1,000), phospho-p53(Ser15) (1:500; Cell Signaling Technology/New England Biolabs), tet-Repressor (1:5,000; MoBiTec/Invitrogen), phospho-Histone H3(S10) (1:1,000; Millipore), and phospho-Cdk1 (T14/Y15) (1:1,000; Santa Cruz), which were obtained from commercial sources. HRP-conjugated secondary antibodies (1:5,000) were from Santa Cruz; conjugated antibodies to FITC, CY3 and CY5 (1:1,000) were obtained from Jackson Immuno Research Labs; and biotinylated secondary antibodies were from DAKO.
Cell proliferation assay
Cell viability and proliferation assays were conducted using the CellTiter-Blue Cell Viability Assay (Promega). Cells were seeded in 96-well plates and siRNA transfected or BI 2536 treated, and after 0, 24, 48, 72 or 96 h, the fluorescence was measured using a Victor 1420 Multilabel Counter (Wallac/PerkinElmer).
The mean mRNA levels measured by qRT-PCR were calculated using the averages of at least five independent samples. All other experiments were performed at least in triplicate. Mouse data depicted in Supplementary Table S2 and S5
were determined by two-way ANOVA, all other assays were analysed by Student's t
-test, unpaired and two-tailed. Significant differences are indicated with stars (*P
Tumour mouse model
Each flank of 8-week-old female athymic mice (Harlan-Winkelmann) was inoculated subcutaneously with 2×106 ES cells resuspended in 0.25 ml sterile saline solution. Luc-iKD ES cells were inoculated on the left flank, whereas Plk1-iKD ES cells were inoculated on the right flank. Mice were distributed randomly into two groups (12 animals each) for the subsequent analysis of tumour development with and without Dox-treatment. Dox (Sigma) was applied as 0.2% solution in the drinking water with additional 3% sucrose dissolved in natural mineral water starting 2 days after inoculation. Drinking water for control mice contained 3% sucrose without Dox. Tumours (teratocarcinomas) were measured in two right-angled dimensions by a sliding caliper three times a week. Absolute tumour volumes were evaluated according to V (mm3)=π/6 ab2 with a>b. After inoculation measurements started at day 7 and finished at day 38 followed immediately by killing the animals. Three individual tumours from all four groups (Luc-iKD- and Plk1-iKD-ES cells, ±Dox) were analysed. The use of animals complied with the regulations of the Goethe-University.
Specific reagents and methodology for the inducible shRNA expression vector (exchange vector), generation of ES cells, mice, analysis of mouse genotypes, stem–loop RT-PCR, western blot, pathology screen, immunohistochemistry, collection and analysis of blood samples are all described in the Supplementary Methods