Supplementary Figures and Tables - Inflammatory Cytokines Rewire the Proinsulin Interaction in Human Islets - Tran et. al. 2022

<strong>Supplemental Figure 1.</strong> <strong>Protein levels for proinsulin and controls in lysate and media.</strong> A) Western blot analysis of expression in lysate and media; Vinculin, Indoleamine 2,3-Dioxygenase 1 (IDO1) and Guanylate Binding Protein 5 (GBP5) and proinsulin. Inset is second Western blot for proinsulin in media samples B) Intracellular proinsulin measured by ELISA and normalized to lysate GAPDH. <br> <strong>Supplemental Figure 2. Proinsulin IP analysis by SDS-PAGE and MS/MS</strong> A) Western blot and silver stain of proinsulin immunoprecipitations from human islets +/- cytokines. B) Total MS/MS counts from proinsulin AP/MS (blue) and IgG AP-MS of six human islets +/- cytokines. C) The total number of proteins identified by AP-MS in distinct islet samples labeled Exp. 6,7,10,13,14,15. For each islet sample there are four IP conditions: Untreated/proinsulin IP, Untreated/IgG IP, Cytokine treated/proinsulin IP, and Cytokine treated/IgG IP. Note that biological samples 13-15 each have two technical replicate MS runs each (R1 and R2). <br> <strong>Supplemental Figure 3. AP-Western validations for selected Proinsulin Interactors identified by AP-MS. </strong>Human islet lysates were immunoprecipitated with antibodies to proinsulin (or IgG), followed by Western blot for ERGIC1, Ataxin-2, ERDJ3, QSOX1, Calnexin, BiP and proinsulin. Men=Menadione, Cyto=Cytokines, M=Markers. <br> <strong>Supplemental Figure 4. Western blot for phospho-eIF2a and total eIF2a. </strong>Human islets, untreated (CON) or cytokine treated (CYTO) were immunoblotted for eIF2a phosphorylated at Ser51 (peIF2 a) and for total eIF2a. MIN6 cells treated with Thapsigargin (Tg) were used as a positive control for peIF2a. GAPDH served as a loading control. <br> <strong>Supplemental Figure 5. KIF2A versus Insulin expression in single human </strong>b<strong>-cells. </strong>For single-cell RNA-Seq data obtained from Gene Expression Omnibus Repository (accession number GSE83139) the normalized reads for insulin (INS), glucagon (GCG), and KIF2A were log2 transformed. We defined b-cell identity as cells with INS log2 normalized reads &gt;12 and with GCG log2 normalized reads &lt;10. By these criteria, 99 cells were chosen for visualization of plotted INS values overlaid with KIF2A values.

**补充图1.** **裂解液与培养基中胰岛素原及对照的蛋白水平。** A) 对裂解液与培养基中的蛋白表达进行免疫印迹（Western blot）分析，检测靶点包括黏着斑蛋白（Vinculin）、吲哚胺2,3-双加氧酶1（Indoleamine 2,3-Dioxygenase 1，IDO1）、鸟苷酸结合蛋白5（Guanylate Binding Protein 5，GBP5）及胰岛素原。内插图为培养基样本中胰岛素原的第二次免疫印迹结果。B) 通过酶联免疫吸附实验（ELISA）检测细胞内胰岛素原水平，并以裂解液中的GAPDH作为内参进行归一化。

**补充图2. 基于SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）与串联质谱（MS/MS）的胰岛素原免疫沉淀分析** A) 来自经细胞因子处理或未处理的人胰岛的胰岛素原免疫沉淀产物的免疫印迹与银染结果。B) 来自经细胞因子处理或未处理的6份人胰岛样本的胰岛素原亲和纯化质谱（AP-MS）总MS/MS计数（蓝色柱）与IgG亲和纯化质谱计数。C) 编号为Exp.6、7、10、13、14、15的不同胰岛样本通过亲和纯化质谱鉴定的总蛋白数。每份胰岛样本包含4种免疫沉淀条件：未处理/胰岛素原免疫沉淀、未处理/IgG免疫沉淀、细胞因子处理/胰岛素原免疫沉淀、细胞因子处理/IgG免疫沉淀。注：生物学样本13-15各包含2次技术重复质谱运行（R1与R2）。

**补充图3. 基于亲和纯化质谱鉴定的选定胰岛素原互作蛋白的亲和纯化-免疫印迹（AP-Western）验证。** 采用抗胰岛素原抗体（或IgG对照）对人胰岛裂解液进行免疫沉淀，随后对ERGIC1、Ataxin-2、ERDJ3、QSOX1、Calnexin、BiP及胰岛素原进行免疫印迹检测。Men=甲萘醌（Menadione），Cyto=细胞因子（Cytokines），M=分子量标志物（Markers）。

**补充图4. 磷酸化eIF2α与总eIF2α的免疫印迹分析。** 将未经处理（CON）或经细胞因子处理（CYTO）的人胰岛，以及用毒胡萝卜素（Thapsigargin，Tg）处理的MIN6细胞（作为磷酸化eIF2α检测的阳性对照）进行免疫印迹，检测Ser51位点磷酸化的eIF2α（peIF2α）与总eIF2α。GAPDH作为上样内参。

**补充图5. 单个成人胰岛β细胞中KIF2A与胰岛素的表达分析。** 从基因表达综合数据库（Gene Expression Omnibus Repository，GEO）获取的单细胞RNA测序数据（登录号GSE83139）中，对胰岛素（INS）、胰高血糖素（GCG）与KIF2A的标准化读取数进行log2转换。我们将β细胞身份定义为：INS的log2标准化读取数>12且GCG的log2标准化读取数<10的细胞。按照该标准，共筛选出99个细胞，用于绘制INS数值与KIF2A数值的叠加散点可视化图。