However, survivin is also found in the beta cells in areas of chronic pancreatitis and within lobular areas of surviving islets of pancreata of patients with presumed type 1A (immune-mediated) childhood-onset diabetes [18,19]. We previously showed that transient perinatal expression of survivin is essential for pancreatic beta-cell mass establishment by regulation of cell cycle progression [20]. Specific deletion of survivin in beta cells does not affect embryonic pancreas or beta-cell development but does impair beta-cell mass expansion, resulting in progressive diabetes in adult male mice [20,21]. However, female mice remain glucose tolerant despite relatively reduced beta-cell mass and thus are the focus of this study, allowing for analysis of adult beta-cell expansion without the confounding effects of pre-existing hyperglycemia. To determine whether survivin is required for beta-cell expansion in adults, we performed partial pancreatic duct ligation (PDL) on adult female RIPCre+survivinfl/fl mice and control (RIPCre+survivin+/+) littermates. Here, we show that survivin, which was absent in adult pancreas was induced within beta cells in the ligated tail of pancreas following PDL during the period of peak regeneration, and that expansion of beta-cell mass following PDL was impaired in RIP-Cre+survivinfl/fl mice due to reduced beta-cell proliferation. Methods Mice Survivinfl/fl mice [12], were mated with mice carrying the Cre transgene under the control of the rat insulin 2 promoter [TgN(ins2-cre)25 Mgn, hereafter referred to as RIPcre; Jackson Laboratories]. RIPcre+survivin+/fl mice were intercrossed to generate RIPcre+survivin+/+, RIPcre+survivin+/fl and RIPcre+sur- vivinfl/fl mice. Only wild-type (RIPcre+survivin+/+) littermates were used as controls. Genotypes for the cre and survivin genes were determined by PCR using mouse tail DNA. PCR primers for survivin (GeneID:11799) were as follows: forward 59- TGAGTCGTCTTGGCGGAGGTTGT-39, reverse 59- GCTCGTTCTCGGTAGGGCAGTGG-39. All mice were main- tained on C57BL/6 background, housed in a pathogen-free facility on a 12-hr light-dark cycle, and fed ad libitum with standard irradiated rodent chow in accordance with Ontario Cancer Institute Animal Care Facility Protocol, without restriction on animal activity. Animal care and experimental protocols were approved by the Nanjing Medical University Animal Care Committee. Surgical Procedure and Tissue Preparation Two-month-old female mice were anesthetized using isoflurane, and laparotomy was performed through a midline abdominal incision. Pancreatic ducts of the gastric and splenic segments were ligated as described [22]. Partial PDL was carefully made from the point where the pancreas is attached to the first portion of the duodenum to the point just before its attachment to the transverse colon. The ligated tail portion accounts for 50–60% of the entire pancreas. For sham operations, the pancreas was gently touched with a moistened cotton-tipped swab for 1 min. Pancreatic head and tail portions were harvested at 3, 7 and 14 days of post- surgery. These specimens were processed separately for weight determination, prior to fixation and embedding in paraffin. Metabolic Studies and Hormone Measurements All overnight-fasts were carried out between 6 pm to 8 am. Blood glucose levels were determined with mouse tail venous blood using an automated glucometer (Roche, Germany). Intraperitoneal glucose tolerance tests (IPGTT) and glucose- Survivin Regulation of Beta-Cell Mass Expansion stimulated insulin secretion (GSIS) measurements were per- formed at day 7 post-surgery as previously described [23]. Serum insulin levels were measured by ELISA with rat/mouse insulin standards (Linco, USA). Islet Isolation Pancreatic islets were isolated from 2-week-old mice and ligated tail portion of the mice at day 7 post-surgeryby injecting collagenase V (2 mg/ml; Sigma-Aldrich, USA) into the pancreas. Pancreata was then allowed to digest in the collagenase solution at 37uC with shaking for 15–20 min. Digestion was stopped by ice- cold Hanks’ balanced salt solution (HBSS) containing 10% (vol./ vol.) FCS. Islets were then hand-picked under a dissecting microscope. Western Blotting Islet-, liver- and kidney-protein lysates were obtained as previously described [24]. Lysates were separated by SDS-PAGE and immunoblotted with antibodies against survivin (Novus Biologicals, USA), p21 and caspase-3 (BD Pharmingen, USA), Aurora Bkinase (Abcam, USA), v-akt murine thymoma viral oncogenes (AKT), phosphorylatedAKT (Ser473), cleaved caspase- 3 (Asp175) and glyceraldehyde phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, USA). Densitometric quantification of protein bands was analyzed using Image J software version 1.37. Samples were normalized to GADPH. Immunohistochemistry, Immunofluorescence Staining, and Islet Morphometry Pancreata were fixed in 4% paraformaldehyde. Samples were dehydrated and prepared as paraffin blocks. Serial seven mm-thick pancreatic sections were obtained with at least 3 levels per slide separated by 100–150 mm intervals and stained with hematoxylin and eosin, insulin (DAKO) and survivin. Beta-cell proliferation was assessed by Ki67 (Novus Biologicals, USA) and apoptosis was detected using In Situ Cell Death Detection Kit (Roche, USA). Double immunofluorescence staining was performed for insulin with survivin or glucagon (NovoCastra Laboratories) and slides were examined using a Zeiss inverted fluorescent microscope. Total beta-cell mass at two month of age and day 7 after PDL were determined on insulin-immunostained sections and calculat- ed by multiplying the pancreatic weight by the ratio of beta-cell area to total pancreatic area. Islet number and beta-cell number and size were calculated on insulin-immunostained sections. According to the nucleus numbers, islets were divided into small islets (1–10 nuclei) and larger islets (.10 nuclei). Quantitative Real-time PCR Total RNA was isolated from ligated pancreas tail at day 7 post- surgery with TRIzol reagent (Invitrogen Life Technologies Inc.,Carlsbad, CA, USA). The cDNA was synthesized from 1 mg RNA using M-MLV reverse transcription system (Promega, Madison, WI, USA). Primers used are listed in Table S1. Real- time RT-PCR analyses were performed using SYBR Green Supermix kit in ABI Prism 7900 HT real-time PCR system (Applied Biosystems, Foster City, CA, USA). The relative levels of gene expression were normalized by the internal control GAPDH. Each sample was run in triplicate, and each experiment included three nontemplate control wells. Statistical Analysis Data are presented as means 6 SE and were analyzed by unpaired student’s t-test and one-way analysis of variance with the PLoS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e41976