distributed on a total of three dishes of 10 cm diameter and incubated in DMEM plus 10% FBS. After 2 weeks of incubation with regular changes of medium every 3 days, dishes were stained with Giemsa, and the number of visible colonies (.1.5 mm of diameter) was scored. For foci formation, early-passage MEFs (P0-P1) derived from individual embryos were seeded (106 cells) in plates of 10 cm diameter and cultured overnight in complete medium containing 10% FBS. The medium was changed 1 h before transfections began. For these assays, we used oncogenic H-rasV12 under an LTR promoter in vector pBabe-puro. Transfections were done by a standard calcium-phosphate procedure with 10 mg of pBabe- puro-H-rasV12 plus 20 mg of carrier DNA plasmid (empty pBabe-puro). After 8 h of incubation with the precipitates, the incubation medium was changed and, 18 h later, cells were distributed equally into three 10 cm diameter plates. Dishes were refed with fresh medium every 3 days. At day 21 post-transfection, cells were fixed and stained with Giemsa, and the total number of foci in the three plates was scored. For retroviral transduction, plasmids indicated in each exper- iment were co-transfected along with pCL-Eco (to produce the ecotropic variant of the Moloney murine leukemia virus) into 293T cells using Fugene 6 (Roche) as reagent, following the manufacturer’s instructions. After two days the supernatant containing the viral particles was filtered by 45 mm pore-filters, mixed with polybrene to a final concentration of 4mg/ml and added to 10 cm diameter plates with the recipient MEFs. This process was repeated three more times, after which infected MEFs were cultured for 2 days in medium with 2 mg/ml puromycin for selection of the infected cells. For induction of apoptosis, cells were either washed twice with PBS and irradiated with a UVC irradiator (Hoeffer) or transduced with viral E1A and subjected to low serum concentration or 0.2 or 0.05 mg/ml adriamycin for two days, after which they were processed for FACS analysis. FACS Analysis Cells were trypsinized and resuspended in cold PBS. Cold ethanol was added dropwise under constant agitation to this cell suspension until reaching a 70% final concentration, and the preparation was kept at 4uC for one to 7 days. These cells were resuspended in PBS containing 30 mg/ml of propidium iodide (Sigma) and 0.1 mg/ml RNase (Invitrogen), incubated at 37uC for 30 minutes, and analyzed by flow cytometry using FACScalibur (BD Biosciences). CellQuest Pro (BD Biosciences) software was used to measure subG0 apoptotic bodies, and the Modfit (Verity Software House) software to obtain cell cycle profiles. Immunoprecipitations and Kinase Assays Protein lysates obtained with the NP-40 buffer (0.5% NP-40, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM phosphate buffer pH 7.2) were pre-cleared by incubating them with protein A or G (Santa Cruz). Supernatants were incubated O/N at 4uC with 5–10 mg antibodies against cyclin D1, cyclin D2 or Cdk2 using orbital agitation, after which protein A or G was added and the mix was incubated at 4uC for 2 hours. Supernatant was kept for the loading control, and the beads were washed three times with the NP-40 buffer. For the IPs, the beads were resuspended in 50 ml of Laemmli buffer, boiled for 5 minutes and immunoblotted using an antibody against Cdk4. For the Cdk2 kinase assays, the beads were washed three times with the kinase assay buffer (20 mM Tris-HCl pH 8; 10 mM MgCl2; 1 mM EGTA; 1 mM DTT; 1 mM PMSF; 1 mM Na3VO4; 1 mM NaF Sei1 in Cancer and Diabetes and a cocktail of protease inhibitors (Roche)). The beads were then incubated with a mix of 20 ml of the kinase buffer, 5 mg of histone H1 (Boeringer), 1 ml of 0.5 mM cold ATP and 10 mCi of c32P-ATP at 30uC for 30 minutes, mixed with 100 ml of Laemmli loading buffer, loaded into a gel and developed by autoradiography. Quantitative RT-PCR Quantitative PCRs of the indicated genes was performed using the 7900Fast qRTPCR system (Applied Biosystems), and data were analyzed using the SDS software provided with it. The oligonucleotides used were the following (listed 59 -.39): Sertad1: sense, CAAGCGGGAGGAGGAGGAGACGAT; antisense, AG- AAGGGGCTGGGGGCTGGAT; Sertad2: sense, CGCACA- GATGACTCACGATT; antisense, GGGGTCAAAATCGTA- CATGG; Sertad3: sense, CAGGCTTGTCGGAGGTAGTC; antisense, TTGGACTCCAGTCCCACTTC; Kir6.2: sense, AGAATATCGTCGGGCTGATG; antisense, CTCTTTCGGA- GGTCCCCTAC; actin, sense, GGCACCACACCTTCTACA- ATG; antisense, GTGGTGGTGAAGCTGTAGCC. Chemical Carcinogenesis For induction of fibrosarcomas, mice of 3–5 months received 1 mg of 3-methyl-cholanthrene (Sigma-Aldrich) in 50 ml of sesame oil by intramuscular injection in the right leg. Mice were examined twice per week until the tumor was palpable and scored in Kaplan- Meier graphs. For induction of papillomas, mice of 2–4 months received a single dose of 20 mg DMBA dissolved in 200 ml of acetone in their shaved backs. Five days later started the treatment with 12.5 mg of TPA dissolved in 200 ml of acetone twice a week for 19 weeks. The number of papillomas was counted, and their size measured with a Vermier caliber during 34 weeks. Glucose Homeostasis and Analysis of Pancreatic Islets To measure pancreatic islets, paraffin sections of pancreas were stained with an insulin antibody (DAKO), and the surface ratios of b-pancreatic islets versus total pancreas were measured using a Mirax scan, and analyzed using the AxioVision software, both from Zeiss. To measure glucose levels, blood samples from male mice fed ad libitum were analyzed for their glucose content using Glucocard Memory 2 Base (A. Menarini Diagnostics). For the high fat diet, mice were fed with Research Diets 12451, 45% kJ from fat, during two months. For the glucose tolerance test (GTT), insulin tolerance test (ITT) and insulin release (IR) assays, groups of wt or Sei1-null males (n = 8 per genotype) of 9 weeks of age were used and all the assays begun after overnight starvation. For the GTT, mice were injected intraperitoneally with a 50% solution of dextrose in PBS and a total amount of 2 g of dextrose per Kg of mouse weight, and blood glucose was measured at 0, 15, 30, 60, 90 and 120 minutes. For the ITT assay, mice were injected with 0.75 U/Kg insulin in saline, and blood glucose was measured at 0, 15, 30, 45, 60 and 90 minutes. For the IR assay, mice were injected with 3 g/Kg dextrose in PBS, and blood samples were obtained at 0, 2, 5, 15 and 30 minutes. Serum from these samples were analyzed for their insulin content using the Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem Inc) and blood glucose was also monitored to control that the assay was correctly performed. Pancreatic islets were isolated following standard methods, essentially as described in [32]. PLoS ONE | www.plosone.org 10 January 2010 | Volume 5 | Issue 1 | e8744