5(c)C5(e)] (21). paramount. A major challenge to expanding cells. Consequently, developing a precise understanding of the molecular pathways that restrain and developed a CC-401 derivative (STF-200785) with improved replication-promoting activity. Finally, we highlight additional cells per well was determined using the Cellomics ArrayScan VTI to image the entire well and count the number of DAPI+PDX1+? or DAPI+insulin+ cells (n = 4 to 8 per condition). Human islets (from 10 donors) of high purity (90% to 95%) and viability ( 87%) from nondiabetic donors (aged 25 to 62 years; median age, 50 years) were obtained through the National Disease Research Interchange and Integrated Islet Distribution Program. Donors were of mixed race (n = 5 white, n = 1 Hispanic, and n = 4 Orlistat black) and sex (n = 5 male, n = 5 Orlistat female); donors were generally obese (average body mass index standard deviation, 30.1 5 kg/m2). cells that coexpressed BrdU were performed by investigators blinded to the treatment cohort. A minimum of 2000 cells from nonconsecutive sections ( 50 m apart) were used to determine the gene as the endogenous reference (22). The primers used in qPCR are listed in Supplemental Material. Expression constructs and luciferase assay Constructs encoding human NFATc1 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_001278669″,”term_id”:”1677499023″,”term_text”:”NM_001278669″NM_001278669) and human DYRK1A (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_001396″,”term_id”:”1889684636″,”term_text”:”NM_001396″NM_001396) were generated and sequence confirmed. The interleukin 2Cbased pGL3-NFAT luciferase reporter construct was obtained from Addgene (catalog no. 17870; Cambridge, MA). Luciferase assays were performed by transfecting (0.625 g polyethylenimine/1 g of DNA) 10-cm tissue culture plates of 90% confluent HEK 293T (RRID: CVCL_0063) cells with DYRK1A (7.5 g), plasmid (10.5 g; Promega), PGL3-NFAT luciferase (4.5 g), and NFATc1 (1.5 g). The next day, cells were trypsinized and transferred to 96-well plates (50 L/well, 1/300th of total cell volume). After 6 hours, wells were treated with vehicle or compound Orlistat as indicated (n = 4 per treatment condition) for 24 hours before being lysed (catalog no. E1500; Promega) for luciferase measurement (Modulus Microplate; Turner Biosystems/Promega). Statistical analysis Statistically significant differences between treatment conditions were determined using the Student two-tailed test; 0.5 was taken to be significant. Experimental results were confirmed in independent experimentation in all cases except for the primary cells were identified by PDX1 expression, also expressed by less prevalent cells, and replication events by Ki67 expression (23). Hit compounds were defined by a twofold increase in cells (n = 4 to 8 replicates per condition; mean SD shown). All compounds increased 0.01). All compound combinations increased 0.01). (f) Rat islet 0.01. Individual data points represent 2000 to 3000 cells (n = 4 to 8 replicates per condition; mean SD shown). Error bars represent the standard deviation of an experimental condition (n 3). cAMP, cyclic adenosine monophosphate; CAS#, Chemical Abstracts Service number; PDE, phosphodiesterase. The purported activities of confirmed replication-inducing compounds clustered into several functional categories. Among the hit compounds were established (GSK-3cells, we tested whether combinations of hit compounds could be used to cooperatively enhance inhibitor CHIR99021 demonstrated inconsistent human inhibitor, CHIR99021, compound combinations that synergistically promoted rat 0.05. Similar data were obtained from at least five independent islet procurements. (c) Rat 0.01; 1000 cells counted per data point. (d) Human 0.05; 1000 cells counted per data point. (e) Representative images of pancreatic sections from 8-week female vehicle- and CC-401Ctreated mice stained for insulin (red), BrdU (green), and nuclei (blue). See Supplemental Fig. 2 for determination of CC-401s half-life and potency. (f) The BrdU incorporation index (percentage of replication) of cells (insulin+) and nonCcells (insulin?) after treatment with vehicle or CC-401 (25 mg/kg) for 1 week. Data from individual mice (n = 5) and mean SD shown. * 0.05. Error bars represent the standard deviation of an experimental condition (n 3). PSFL Two independent experiments were performed with similar results. See Supplemental Fig. 2 for replication effects on cells, cells, and dermal fibroblasts. ALKV Inh..