REST Target Genes and Beta Cell Survival Figure 1. Diabetic RIP-REST transgenic mice are hyperglycemic and show poor insulin secretion. A. Blood glucose levels were assessed at different ages in male and female diabetic RIP-REST transgenic (dark diamonds; n = 7) and wild type mice (open diamonds; n = 5). Diabetic RIP-REST mice feature hyperglycemia from weaning onward. Results are mean 6 SD. ***P,0.001 versus values of wild type mice. B. Diabetic RIP-REST mice (black circles, n = 3) and wild type littermates (open squares, n = 4) were subjected to in situ pancreatic perfusion at 1.5 ml/min rate. After a 30-min equilibration period at basal 1.4 mmol/l glucose, the pancreas was perfused sequentially at different glucose concentrations, first at 1.4 mmol/l for 20 min, next at 8.0 mmol/l for 20 min, then at 16.0 mmol/l for 20 min, followed by a 30-min stimulation at 8.0 mmol/l plus 1 nmol/l GLP-1, and finally at 1.4 mmol/l for 15 min. Results are mean 6 SD. doi:10.1371/journal.pone.0045844.g001 (CTNND2) and apoptosis inhibitor 5 (API5) were significantly reduced after expression of REST in INS-1E cells (Fig. 5B). While IRS2 and PTPRN are known to have anti-apoptotic effects [4,10], the latter four proteins were not, prior to our study. To verify whether these genes were downregulated in vivo upon REST expression, and could account for the apoptotic events occurring in the diabetic RIP-REST mice, we analyzed islets of RIP-REST mice. qPCR experiments showed that the expression levels of several REST target genes, including Gjd2, Mapk8ib1, Ptprn and Cdk5r2 were reduced (Fig 5C). In contrast, we did not see any downregulation for Irs2, Nrg1 or Ctnnd2 in these transgenic mice (Fig 5C). Decreased production of Cdk5r2 upon REST expression was also confirmed at the protein level using INS–1E cells transduced with Ad-REST and Ad-GFP as a control (Fig. 5D). We further evaluated the possibility that beta cells from RIP-REST mice were suffering from apoptosis, due to ER stress that may be induced by enhanced levels of RIP-mediated protein translation. However, we failed to detect any increase in the transcript levels of the ER stress markers, Chop, Bip and splice Xbp-1 in the islets of RIP-REST mice, compared to wild type animals (Fig. S1). Specific siRNA silencing of Cdk5r2 increases INS-1E cells susceptibility to cytokines We then assessed the role of Cdk5r2, given that this protein was not yet known to be involved in beta cell apoptosis. Out of three siRNAs tested, we selected two specific rat Cdk5r2 siRNAs (siCdk5r2#1 and siCdk5r2#2) which efficiently silenced the target sequence, as judged by qPCR experiments conducted on transfected INS-1E cells (data not shown). Transfection of these siRNAs into INS-1E cells resulted in a 70% to 80% reduction of Cdk5r2 protein level after 72 h (Fig. 6A). This change did not affect the levels of Cdk5r1 (also called p35), another activator of CDK5 (data not shown). We then evaluated the effect of silencing Cdk5r2 PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45844