Phospho-antibody microarray analyses for islets of control and Mettl14 knock-out mice. Phospho-antibody microarray analyses for islets of control and Mettl14 knock-out mice

β-cell specific Mettl14 knock-out mice display reduced N6-methyladenosine (m6A) levels and recapitulate human Type II diabetes (T2D) islet phenotype with early diabetes onset and mortality secondary to decreased β-cell proliferation and insulin degranulation. To gain insights into the role of m6A in regulating the IGF1/insulin -> AKT - > PDX1 pathway and to dissect the signaling networks modulating AKT phosphorylation, we subjected freshly isolated islets from control and Mettl14 knock-out mice to phospho-antibody microarrays. Overall design: Mettl14fl/fl mice on a C57BL/6N background was created and provided by Dr. Chuan He (University of Chicago). To obtain β-cell specific Mettl14 knock-out mice, Mettl14fl/fl mice were breed with B6(Cg)-Ins1tm1.1(cre)Thor/J (Jackson Labs, USA) and male mice wild-type for nicotinamide nucleotide transhydrogenase (Nnt) mutation were used. Mice were weaned at 3 weeks of age and maintained on a chow diet (PicoLab® mouse diet 20 – 5058). Islet isolations were performed in anesthetized mice and their pancreas infused with liberase (Roche, Germany). Following incubation at 37C for 17 min the digested pancreases were washed filtered through a 400µm filter and run on a Histopaque (Sigma, USA) gradient. The purified islets were handpicked, counted and cultured overnight in 7mM glucose RPMI media (Gibco, USA) containing 10% FBS and 1% PS (Gibco, USA). Islets were handpicked, washed 2 times with ice-cold DPBS by self-sedimentation. Protein lysates from 200 size-matched islets from each sample (4 control pools and 2 Mettl14 knock-out pools) were used for Kinex™ KAM-1325 antibody microarray analyses.

β细胞特异性Mettl14敲除小鼠（β-cell specific Mettl14 knock-out mice）的N6-甲基腺嘌呤（N6-methyladenosine，m6A）水平降低，且可重现人类II型糖尿病（Type II diabetes，T2D）的胰岛表型，表现为早发糖尿病及死亡，该表型继发于β细胞增殖减弱与胰岛素脱颗粒。为探究m6A在调控IGF1/胰岛素→AKT→PDX1通路中的作用，并解析调控AKT磷酸化的信号网络，我们对对照组与Mettl14敲除小鼠的新鲜分离胰岛进行了磷酸化抗体芯片检测。实验整体设计：本研究使用的C57BL/6N背景Mettl14fl/fl小鼠由芝加哥大学Chuan He博士构建并赠予。为获得β细胞特异性Mettl14敲除小鼠，将Mettl14fl/fl小鼠与B6(Cg)-Ins1tm1.1(cre)Thor/J（美国杰克逊实验室）交配，并选用烟酰胺核苷酸转氢酶（nicotinamide nucleotide transhydrogenase，Nnt）突变位点为野生型的雄性小鼠。小鼠于3周龄断奶，采用普通饲料（PicoLab®小鼠饲料20——5058）饲养。胰岛分离操作在麻醉小鼠中进行，通过胰胆管灌注胶原酶Liberase（德国罗氏公司）。37℃孵育17分钟后，将消化的胰腺洗涤，经400μm滤网过滤，随后采用Histopaque（美国Sigma公司）密度梯度离心分离。纯化后的胰岛经手工挑取、计数后，置于含10%胎牛血清（FBS）与1%双抗（PS）的7mM葡萄糖RPMI培养基（美国Gibco公司）中过夜培养。后续再次手工挑取胰岛，采用冰浴DPBS通过自然沉降洗涤2次。每个样本取200个大小均一的胰岛制备蛋白裂解液（对照组共4个混合样本，Mettl14敲除组共2个混合样本），用于Kinex™ KAM-1325抗体芯片分析。