Figure 6. CDK5R2 protects beta cells against cytokines and palmitate. A. Left panel, immunoblotting of total proteins from INS-1E cells, 72 h after transfection with a negative siRNA (siNeg) or siRNAs against Cdk5r2 (siCdk5r2#1 and siCdk5r2#2). Right panel, quantifica- tion of the corresponding Cdk5r2 protein levels. Results are mean 6 SEM of three independent experiments. **P,0.01 versus values of siNeg-transduced INS-1E cells. B. Quantification of apoptotic nuclei in non-transfected INS-1E cells (NT) and INS-1E cells transfected with a negative siRNA (siNeg) or with siRNAs against Cdk5r2 (siCdk5r2#1 and siCdk5r2#2), treated 24 h with a mix of cytokines (black bars) or not (white bars). Results are mean 6 SEM of six independent experiments. **P,0.01 versus controls. C. Immunoblotting of total proteins from INS- 1E cells transfected with a negative siRNA (siNeg) or siRNAs against REST Target Genes and Beta Cell Survival Cdk5r2 (siCdk5r2#1 and siCdk5r2#2), treated 24 h with a mix of cytokines (Cyt) or not (Ctrl). The cleavage of caspase-9 and 23, induced by cytokines, is higher in siCdk5r2- than in siNeg-treated cells. D. Quantification of apoptotic nuclei in non-transfected INS-1E cells (NT) and INS-1E cells transfected with a negative siRNA (siNeg) or siRNAs against Cdk5r2 (siCdk5r2#1 and siCdk5r2#2), exposed 15 h to control medium (white bars) or palmitate (black bars). Results are mean 6 SEM of six independent experiments. **P,0.01 versus controls. doi:10.1371/journal.pone.0045844.g006 an islet) by applying the MaxEntropy thresholding method. After application of the ‘‘open’’ binary operation, particles were detected fixing the minimum size at 100. The average pixel intensity of nuclei (i.e, identified particles), was then measured from the original cropped image and values obtained from at least 10 islets from three animals for each group were subjected to statistical analyses. For cell mass quantification, the relative area occupied by the different types of islet cells were measured on 16 sections taken throughout the entire pancreas and separated by at least 200 mm (adult animals), or 150 mm (newborn animals). Islet cells and total pancreas areas were measured using an ACECAD Professional graphic tablet connected to a Quantimet Leica 5001 (Leica, Cambridge Ltd, England) programmed for semiautomatic mea- surement of areas. Glucose tolerance test Male mice of 12–16 weeks were fasted for 15 h before blood samples were collected from the tail vein at 0 (fasting blood sample), 15, 30 and 120 min after an intraperitoneal injection of glucose (2 g/kg of body weight as a 20% solution). Blood glucose levels were measured with a Glucometer (Bayer AG Health Care, Switzerland). Cell line and mouse islet isolation The rat insulinoma cell line INS-1E was maintained in RPMI 1640 medium, as previously described [13]. For infection experiments, INS-1E cells were seeded in 12-well plates and cultured for 48 h before infection with adenoviruses as previously described [46], with a multiplicity of infection of 2 or 5. Islets of Langerhans of adult C57BL/6 male mice, weighing 25–30 g, were isolated and cultured as previously described [13]. RNA isolation and real time RT-PCR RNA isolation was performed as previously described [47]. Quantitative RT-PCR (qPCR) was performed using the SYBR Premix Ex Taq PCR Kit TaKaRa (Axon Lab, Switzerland) in a Lightcycler (Roche Diagnostics GmbH, Mannheim, Germany), as previously described [47]. cDNAs were amplified using specific primers (Table S1). Apoptosis assay Apoptosis was determined by scoring cells displaying picnotic nuclei stained with the DNA-binding dyes Hoechst 33342 (10 mg/ ml; Molecular probes, Eugene, Oregon, USA), as visualized under an inverted fluorescence microscope. A minimum of 500 cells were counted in each experimental condition and viability was evaluated by two independent observers, one of them unaware of sample identity. Western blotting Western blots were performed as previously described [47]. Specific protein levels were revealed with polyclonal rabbit antibodies against Cdk5r2, cleaved caspase-3 and cleaved PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e45844