includes the mammalian Plks: Plk1, Plk2 (Snk), Plk3 (Fnk/Prk) and Plk4 (Sak-a/b); Xenopus laevis Plx1,[33] Schizosaccharomyces pombe plo1+ and Saccharomyces cerevisiae CDC5 [34–36]. Plk1 is important for mitotic progression (karyokinesis and cytokinesis); it is involved in the nuclear translocation of cyclin B1,[37] microtubule dynamics,[38–42] activation of Cdc25C,[42] inactivation of Wee1[43,44] and Myt1,[45] separation of chro- mosomes at anaphase and destruction of mitotic proteins through activation of the anaphase promoting complex/cyclosome (APC/ C).[46,47] Recently, it has been demonstrated that Plk1 contributes to cytokinesis in human cells[48,49] and that inhibition of Plk1 activity with the highly selective inhibitor, BI 2536,[48,50,51] results in cytokinesis failure and the generation of binucleated cells. Plk2–4 display varying roles in cell cycle progression: Plk2 plays a role in centriole duplication;[52] Plk3 is a non-cycling Plk[53] involved in G2 checkpoint signaling.[54] Plk4 differs from Plk1–3 as it contains a variation of the Plk kinase motif and has a single polo-box.[55] Plk4 has been shown to localize to centrioles and, like Plk2, is thought to be important for centriole duplication. Loss or over expression of Plk4 results in defects in the centriole cycle and aberrant spindle formation [56–59]. Previously, Plk1 mRNA and protein have been shown to decline during rat cardiac myocyte development.[60] We have extended these studies and characterized the expression of the whole family of mammalian Plks during rat cardiac myocyte development. We confirmed that the expression and activity of Plk1 in cardiac myocytes declines during development in accordance with observa- tions previously reported.[60] Plk2, Plk3 and Plk4 also showed small, but significant, changes at the mRNA and protein levels during development. In addition, we have tested the hypothesis that over expression of Plk1 would reinitiate the cell cycle in G2/M-arrested cells and increase the number of cardiac myocytes. Adenovirus constructs were generated to express wild-type Plk1 or a constitu- tively active Plk1 with the phospho-mimicking T210D mutation. Despite being able to increase HeLa cell proliferation, we found that over expression of Plk1 was unable to induce cell cycle progression in isolated neonatal rat cardiac myocytes at 48 hours, or in adult rat cardiac myocytes at 24 hours. We found that this most likely due to the inability of Plk1 to increase the activity of Cdc2/cyclin B1. Based on these findings, we sought to further characterize the G2/M arrest originally observed in adult cardiac myocytes with the aim of determining a more precise description of the G2/M arrest. Using an immunohistochemical approach, we found no evidence of G2/M marker expression in the adult myocardium and conclude that this may be indicative of an earlier arrest. Materials and Methods Ethics Statement Ventricular cardiac myocytes were isolated from wild type Wistar rats which were housed and maintained in a colony in accordance with the Local Ethical Review Panel of the University of Reading and U.K. Home Office guidelines. All experimental procedures using animals were performed in accordance with the U.K. Animals (Scientific Procedures) Act, 1986. Materials Dual-labeled quantitative PCR probes and oligonucleotides were synthesized by Sigma-Genosys (Pampisford, U.K.). ABgene Absolute QPCR Rox Mix (ABgene; Epsom, UK) was used according to the manufacturer’s instructions. Samples were normalized to either the ribosomal subunit, 18S, using the 18S QPCR kit (Applied Biosystems, Warrington, UK) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) QPCR kit (Applied Biosystems, Warrington, UK). Plk1 & Myocardial Regeneration Samples were amplified on an Applied Biosystems 5700 GeneAmp QPCR machine. The High-Fidelity PCR system was supplied by Roche Diagnostics (Lewes, U.K.). Anti-(cyclin B1) (GNS1) and anti- (CDC2 p34), antibodies were purchased from Santa Cruz Biotech- nology (Santa Cruz, CA, U.S.A.). Anti-Plk1 monoclonal antibody cocktail was purchased from Zymed (Invitrogen, Paisley, U.K.). Anti- Plk2 and Plk4 antibodies were purchased from Bethyl Laboratories (Montgomery, TX, U.S.A.), and an anti-Fnk/Plk3 antibody was purchased from BD Biosciences. The anti-tropomyosin (sarcomeric) antibody (clone CH1) was obtained from Sigma (Poole, U.K.). Anti- Histone H3 Ser10 antibody was purchased from Millipore (Watford, U.K.) and anti-Histone H3 Ser28 and Ki67 antibodies were purchased from Abcam (Cambridge, U.K.) Horseradish-peroxi- dase-conjugated secondary antibodies were purchased from Dako- Cytomation (Ely, U.K.) and AlexaFluorH 568 and 488 anti-mouse IgG secondary antibodies were purchased from Molecular Probes (Eugene, OR, U.S.A.). Vectashield mounting medium containing DAPI (49,6-diamidino-2-phenylindole) was purchased from Vector Laboratories (Orton Southgate, Peterborough, U.K.). Expression vector pIRES2-EGFP (enhanced green fluorescent protein) was obtained from BD Clontech UK (Basingstoke, U.K.). Quantity OneH software package was from Bio-Rad (Hemel Hempstead, U.K.). BD MatrigelTM Matrix (growth factor reduced) was supplied by Fahrenheit Laboratories (Milton Keynes, U.K.). All other chemicals and biochemicals were supplied by Sigma. HeLa Cell Proliferation Assay HeLa cells (56105) were plated into 6 well dishes and maintained in DMEM supplemented with 10% FCS for 24 hours prior to infection. Cells were infected at an MOI of 50 and cell number was assessed by Coulter Counter at 48 hours post infection. Cells were washed, trypsinised and resuspended in Isoton II solution (Beckton Dickinson, Oxford, U.K.) prior to counting. Cardiomyocyte isolation Fetal [gestational day 18 (E18)] and neonatal [2- (P2), 3- (P3), 5- (P5), 7- (P7) and 10- (P10) days old] ventricular cardiomyocytes were isolated from 30–70 excised ventricles of Wistar rats as described previously [12,18]. Cells were maintained in either serum-free (0%) myocyte media (80% DMEM, 20% M199) or myocyte media supplemented with 5% fetal calf serum (FCS). Cardiomyocyte-enriched cultures were at least 95% pure, as determined by anti-tropomyosin staining. Adult ventricular cardiomyocytes were isolated from the excised hearts of male Wistar rats (175–200 g) by Langendorff perfusion [10,13] and maintained in modified M199 media (M199 supplemented with 2 mM creatine, 2 mM carnitine and 5 mM taurine). Quantitative PCR Primers and probes were designed using Primer ExpressTM Software (Applied Biosystems, Warrington, UK) as follows: Plk1 Rat Plk1 QPCR-Fwd 1121:59- CTCCCGAACCACTGGTTCC-39, Rat Plk1 QPCR-Rev 1189: 59- CTTAATAAAGGTGTGGAGAACC- CC-39, Rat Plk1 Probe: 59-TAMRATGCCTGACCGTCCCCGGA- GAFAM- 39; Plk2 QPCR-Fwd: 59-GTCCTTTCAGTGGGTCAC- CAA-39 Rat Plk2 QPCR-Rev: 59- GTGTGGTCTGAGAGCTGG- TATCC-39, Rat Plk2 Probe: 59-TAMRATGGGTCGACTACT- CCAACAAATACGGCTT-FAM-39; Plk3 QPCR-Fwd: 59-GCCTA- CGCGGTCAAAGTCA-39, Rat Plk3 QPCR-Rev: 59- TTATGA- TCTTCTCGCGCTGATG-39, Rat Plk3 Probe: 59-TAMRACCG- CAGAGTCGCGTCGCCAFAM- 39; QPCR Rat Plk4-Fwd: 59- AA- GCAGAATGAGCGAGAATAG-39, QPCR Rat Plk4-Rev: 59- CC- TTCCACTGTCACAGCAGAAG- 39, Rat Plk4: 59-TAMRACG- GCTTTCCCAACACAGACACCAGTACTC-FAM-39. PLoS ONE | www.plosone.org 2 August 2009 | Volume 4 | Issue 8 | e6752