Images were acquired on an ImageXpress Micro automated microscope (Molecular Devices)

Images were acquired on an ImageXpress Micro automated microscope (Molecular Devices). cell states in beta-cell reprogramming from alpha cells. These small-molecule studies suggested that inhibition of kinases in particular may induce the expression of several beta-cell markers in alpha cells. To identify potential lineage reprogramming protein targets, we compared the transcriptome, proteome, and phosphoproteome of alpha cells, beta cells, and compound-treated alpha cells. Our phosphoproteomic analysis indicated that two kinases, BRSK1 and CAMKK2, exhibit decreased phosphorylation in beta cells compared to alpha cells, and in compound-treated alpha cells compared to DMSO-treated alpha cells. Knock-down of these kinases in alpha cells resulted in expression of key beta-cell markers. These results provide evidence that perturbation of the kinome may be important for lineage reprogramming of alpha cells to beta cells. Introduction Type-1 diabetes (T1D) is a chronic autoimmune disease affecting 35 million patients worldwide. In T1D, insulin-secreting pancreatic beta cells are destroyed by autoreactive immune cells [1], [2]._ENREF_1 The most common treatment for T1D is daily injection of insulin; however, this treatment cannot always ensure optimal glucose homeostasis, leading to complications such as blindness, heart disease, limb amputation, and ultimately death [3]. Another therapeutic strategy involves transplantation of pancreatic islets [4] via infusion through the portal vein into the liver, but high cost [5], limited donor availability, and beta-cell toxicity [6], [7] of immunosuppressive drugs severely restrict the use of this treatment protocol. Since T1D is characterized by extreme loss of beta-cell mass, replenishing the beta-cell population by converting other pancreatic cell types, such as alpha cells, to beta cells may be a viable therapeutic strategy [8], [9]. For example, lineage reprogramming of pancreatic alpha cells to beta cells by ectopic expression of transcription factor, and Gene Solution siRNAs (Qiagen) were used to perform the gene knock-down experiments. Quantitect primer sets for each gene were used to determine gene expression. Mouse TC1 cells were plated in 96-well Corning (3340) plates at 40,000 cells/well in 100 L DMEM. 0.3 L/well LipofectamineTM RNAiMAX (Invitrogen) and Opti-MEM Media was used to transfect the mix of 4 different siRNA constructs into cells. Cells were incubated in transfection mix for 6 h at 37C, before changing to fresh media. Cells were cultured for 3 days, followed by qPCR or immunohistochemistry. Statistical significance was determined using t-test. shRNA experiments The different shRNA lentiviruses were obtained from the RNAi Consortium (TRC; http://www.broadinstitute.org/rnai/public/) in 96-well format, with approximately 108 viral particles/mL per well. Hairpin identities are listed in Table S1 in File S1. Mouse TC1 cells were plated in 96-well Corning (3340) plates at 40,000 cells/well in 100 L standard culture media. The next day, polybrene was PT2977 added to each well (6 g/mL), and cells were spin-infected with 1 L virus at 2,250 rpm for 30 min at 30C. Media was changed 4 h later to fresh media. The next day, media containing 1 g/mL puromycin was added. Puromycin-supplemented media was changed every 3 days. After 10 days in culture, cells were lysed and mRNA extracted using Qiagen RNeasy 96 Kit. Statistical significance was determined using t-test. Immunocytochemistry Cells were fixed with 4% PFA for 15 min, followed by a 0.2% Triton-X-100 permeabilization for 20 min, and blocking with PBS supplemented with 2% BSA for 2 h. Fixed cells were then incubated with a mix of 1200 rabbit anti-Pdx1 (Abcam) and 1500 guinea pig anti-insulin (Sigma) overnight at 4C. As secondary antibodies, AlexaFluor594 anti-rabbit and AlexaFluor488 (Invitrogen) anti-guinea pig were used. Images were acquired on an ImageXpress Micro automated microscope (Molecular Devices). Exposure settings: 600 ms for Pdx1 (TxRed), 200 ms for insulin (FITC), and 8 PT2977 ms for DAPI. Statistical significance was determined using t-test. Gene expression Total mRNA from siRNA and compound-treated cells were extracted with Qiagen RNeasy Plus Mini Kit. qPCRs were performed with Power SybrGreen PCR Master Mix and an Applied Biosystems 7900HT plate reader. Mouse primers were obtained from IDT and are listed in Table S2 in File S1. Results Experimental strategy and rationale We were interested in comparing the genetic and proteomic levels of various transcription factors in alpha and beta cells, as such proteins are linked with lineage reprogramming. Thus, we compared the gene-expression profiles of the mouse alpha-cell (TC1) and beta-cell (TC3) lines (Figure 1A). In parallel, we compared the proteomes and phosphoproteomes of alpha cells and beta cells using stable isotope labeling by amino acids in Cd63 cell culture (SILAC; Figure 1B) [19]C[21]. We used a three-state labeling strategy, with two states corresponding to the individual cell populations, and the third state corresponding to a PT2977 mixed proteome from alpha and beta cells. This approach enabled.

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