<strong>Response to oxidative stress of AML12 hepatocyte cells with knockout of methionine sulfoxide reductases</strong> Jose Abraham Trujillo-Hernandez and Rodney L. Levine. Published in the journal of Free Radical Biology &amp; Medicine

This is a set of data, published in the journal of Free Radical Biology &amp; Medicine, by Jose Abraham Trujillo-Hernandez and Rodney L. Levine: <br> <strong>Figure 1. AML12 is a hepatocyte-like cell line. (A)</strong> RT-PCR to identify the expression of hepatic genes on AML12 cells. <em>Gapdh</em> gene was used as a positive control. <strong>(B) </strong>Western blot for MSRA protein expression on AML12 cells. Recombinant MSRA and mouse liver extract were used as positive controls, and negative controls consisted of liver extract from the quadruple knockout mouse and HEK293 cell extract. <strong>Figure 2. Depletion of methionine sulfoxide reductase in AML12 cells. (A)</strong> PCR genotyping of the knockout cell lines. The lower band characterizes the homozygous knockout line for each gene resulting from a partial deletion within the gene. The heterozygous line <em>Msra+/-</em> is identified by a double band pattern. WT = wild type, <em>Msrb3KO -Msra+/- </em>= heterozygous <em>Msra</em> in the triple knockout of the Msrb family (<em>Msrb3KO</em>), and homozygous <em>Msra</em> = <em>Msra -/-</em>. Western blot in <strong>(B)</strong> and chart in <strong>(C)</strong> from panel (B) show the quantification of MSRA protein content in the knockout lines (Four independent repetitions). We used recombinant MSRA as a reference to calculate the amount of protein in the AML12 lines. Student's t-test was used to determine significant differences. * = P-value equal or less than 0.01, ** = P-value equal or less than 0.001. <strong>Figure 3.</strong> <strong>Ischemia-reperfusion injury of AML12 cells.</strong> <strong>(A) </strong>Experimental procedure to mimic ischemia-reperfusion (red upper panel). Damage is measured by cell viability with tetrazolium salt (blue lower panel). <strong>(B)</strong> Effect of 24 h serum, glucose, and oxygen deprivation. <strong>(C)</strong> Effect of glucose and oxygen deprivation for 36 h ischemia followed by 3 h reperfusion. * = P-value equal or less than 0.01, ** = P-value equal or less than 0.001, (Three independent repetitions). <strong>Figure 4. MSR knockout cells response to oxidative stress.</strong> <strong>(A)</strong> Cell viability after 36 h ischemia and 3 h reperfusion (Three independent repetitions). <strong>(B) </strong>Cell viability after two days incubation with 90 µM paraquat (Three independent repetitions). There were no significant differences among the cell lines. <strong>Supplementary figure 1. Generation of MSR knockout lines. (A)</strong>, <strong>(C)</strong>, <strong>(E)</strong>, and <strong>(G)</strong> show the strategy to knockout each gene of the MSR family. Red scissors indicate CRISPR sites. <strong>(B)</strong>, <strong>(D)</strong>, <strong>(F)</strong>, and <strong>(H)</strong> show the PCR-based genotyping of the homozygous lines that are characterized by the lower band. <strong>(I)</strong> Western blot confirms depletion of MSRA by CRISPR/Cas9 in the <em>Msra</em>-/- knockout line. WT mouse liver, WT AML12, and recombinant MSRA were used as positive controls. Mouse liver from the quadruple mouse was used as a negative control. <strong>Supplementary figure 2. The response of individual Msrb knockout lines to oxidative stress. (A) Cell viability after 36 h ischemia and 3 h reperfusion </strong>(Three independent repetitions). <strong>(B) Dose response to paraquat of WT AML12 cells </strong>(Three independent repetitions). <strong>(C) Cell viability after 2 days incubation with 90 uM paraquat </strong>(Four independent repetitions)<strong>.</strong> Student's t-test was used to determine significant differences. * = P-value equal or less than 0.01, ** = P-value equal or less than 0.001.

本数据集由Jose Abraham Trujillo-Hernandez与Rodney L. Levine发表于《自由基生物学与医学（Free Radical Biology & Medicine）》期刊：<br> <strong>图1 AML12为类肝细胞系。(A)</strong> 采用逆转录聚合酶链反应（Reverse Transcription Polymerase Chain Reaction, RT-PCR）鉴定AML12细胞的肝脏基因表达，以<em>Gapdh</em>基因作为阳性对照。<strong>(B)</strong> 采用蛋白质免疫印迹（Western blot）检测AML12细胞中MSRA蛋白的表达；以重组MSRA与小鼠肝脏提取物作为阳性对照，以四基因敲除小鼠肝脏提取物及HEK293细胞（Human Embryonic Kidney 293 Cell, HEK293）提取物作为阴性对照。<br> <strong>图2 甲硫氨酸亚砜还原酶（Methionine Sulfoxide Reductase, MSR）敲除的AML12细胞构建。(A)</strong> 敲除细胞系的PCR基因分型：下方条带代表对应基因内部部分缺失产生的纯合子敲除株；杂合子<em>Msra+/-</em>呈现双条带模式。WT为野生型，<em>Msrb3KO -Msra+/-</em>代表MSRB家族三敲除（<em>Msrb3KO</em>）背景下的<em>Msra</em>杂合子，纯合子<em>Msra</em>即为<em>Msra-/-</em>。<strong>(B)</strong> 的蛋白质免疫印迹结果与<strong>(C)</strong> 的定量图表展示了敲除细胞系中MSRA蛋白含量的定量分析（共4次独立重复实验）；以重组MSRA作为参考标准计算AML12细胞系中的蛋白丰度，采用学生t检验确定显著性差异，*表示P值≤0.01，**表示P值≤0.001。<br> <strong>图3 AML12细胞的缺血再灌注损伤模型。(A)</strong> 模拟缺血再灌注的实验流程（红色上半部分）；通过四氮唑盐（Tetrazolium Salt）法检测细胞活力以评估损伤程度（蓝色下半部分）。<strong>(B)</strong> 24小时血清、葡萄糖及氧剥夺的影响。<strong>(C)</strong> 36小时葡萄糖与氧剥夺后再灌注3小时的影响。*表示P值≤0.01，**表示P值≤0.001（共3次独立重复实验）。<br> <strong>图4 MSR敲除细胞对氧化应激的响应。(A)</strong> 36小时缺血及3小时再灌注后的细胞活力（共3次独立重复实验）。<strong>(B)</strong> 经90 µM百草枯（Paraquat）孵育2天后的细胞活力（共3次独立重复实验）；各细胞系间无显著性差异。<br> <strong>补充图1 MSR敲除细胞系的构建。(A)</strong>、<strong>(C)</strong>、<strong>(E)</strong>、<strong>(G)</strong>展示了MSR家族各基因的敲除策略，红色剪刀代表成簇规律间隔短回文重复序列相关蛋白9（Clustered Regularly Interspaced Short Palindromic Repeats Associated Protein 9, CRISPR/Cas9）的靶点位点。<strong>(B)</strong>、<strong>(D)</strong>、<strong>(F)</strong>、<strong>(H)</strong>展示了以下方条带为特征的纯合子敲除株的PCR基因分型结果。<strong>(I)</strong> 蛋白质免疫印迹验证了CRISPR/Cas9介导的<em>Msra-/-</em>敲除细胞系中MSRA的敲除效果；以野生型小鼠肝脏、野生型AML12细胞及重组MSRA作为阳性对照，以四基因敲除小鼠肝脏提取物作为阴性对照。<br> <strong>补充图2 单个<em>Msrb</em>敲除细胞系对氧化应激的响应。(A) 36小时缺血及3小时再灌注后的细胞活力</strong>（共3次独立重复实验）。<strong>(B) 野生型AML12细胞对百草枯的剂量响应曲线</strong>（共3次独立重复实验）。<strong>(C) 经90 µM百草枯孵育2天后的细胞活力</strong>（共4次独立重复实验）。采用学生t检验确定显著性差异，*表示P值≤0.01，**表示P值≤0.001。