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

This is a set of data, published in the journal of Free Radical Biology & Medicine, by Jose Abraham Trujillo-Hernandez and Rodney L. Levine: Figure 1. AML12 is a hepatocyte-like cell line. (A) RT-PCR to identify the expression of hepatic genes on AML12 cells. Gapdh gene was used as a positive control. (B) 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. Figure 2. Depletion of methionine sulfoxide reductase in AML12 cells. (A) 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 Msra+/- is identified by a double band pattern. WT = wild type, Msrb3KO -Msra+/- = heterozygous Msra in the triple knockout of the Msrb family (Msrb3KO), and homozygous Msra = Msra -/-. Western blot in (B) and chart in (C) 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. Figure 3. Ischemia-reperfusion injury of AML12 cells. (A) Experimental procedure to mimic ischemia-reperfusion (red upper panel). Damage is measured by cell viability with tetrazolium salt (blue lower panel). (B) Effect of 24 h serum, glucose, and oxygen deprivation. (C) 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). Figure 4. MSR knockout cells response to oxidative stress. (A) Cell viability after 36 h ischemia and 3 h reperfusion (Three independent repetitions). (B) Cell viability after two days incubation with 90 µM paraquat (Three independent repetitions). There were no significant differences among the cell lines. Supplementary figure 1. Generation of MSR knockout lines. (A), (C), (E), and (G) show the strategy to knockout each gene of the MSR family. Red scissors indicate CRISPR sites. (B), (D), (F), and (H) show the PCR-based genotyping of the homozygous lines that are characterized by the lower band. (I) Western blot confirms depletion of MSRA by CRISPR/Cas9 in the Msra-/- 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. Supplementary figure 2. The response of individual Msrb knockout lines to oxidative stress. (A) Cell viability after 36 h ischemia and 3 h reperfusion (Three independent repetitions). (B) Dose response to paraquat of WT AML12 cells (Three independent repetitions). (C) Cell viability after 2 days incubation with 90 uM paraquat (Four independent repetitions). 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.

本数据集由何塞·亚伯拉罕·特鲁希略-埃尔南德斯（Jose Abraham Trujillo-Hernandez）与罗德尼·L·莱文（Rodney L. Levine）发表于《自由基生物学与医学》（Free Radical Biology & Medicine）期刊，具体实验内容如下：

图1：AML12为类肝细胞系（hepatocyte-like cell line）。(A) 采用逆转录聚合酶链反应（RT-PCR）鉴定AML12细胞中肝脏相关基因的表达情况，以Gapdh基因作为阳性对照。(B) 采用蛋白质印迹法（Western blot）检测AML12细胞中甲硫氨酸亚砜还原酶A（MSRA）的蛋白表达水平，以重组MSRA与小鼠肝脏提取物作为阳性对照，阴性对照则为四基因敲除小鼠肝脏提取物与人胚胎肾细胞293株（HEK293）细胞提取物。

图2：AML12细胞中甲硫氨酸亚砜还原酶的敲除。(A) 敲除细胞系的PCR基因分型：低分子量条带代表对应基因内部部分缺失形成的纯合敲除细胞系；杂合细胞系Msra+/-呈现双条带模式。WT为野生型，Msrb3KO -Msra+/-代表甲硫氨酸亚砜还原酶B（Msrb）家族三敲除（Msrb3KO）背景下Msra基因的杂合敲除，纯合Msra敲除细胞系记为Msra -/-。(B) 的蛋白质印迹结果与(C) 的定量图表展示了各敲除细胞系中MSRA蛋白含量的定量分析（共4次独立重复实验），以重组MSRA作为参照计算各AML12细胞系中的蛋白含量。采用学生t检验确定显著性差异：*代表P值≤0.01，**代表P值≤0.001。

图3：AML12细胞的缺血再灌注损伤模型。(A) 模拟缺血再灌注的实验流程（红色上方面板），通过四唑盐法检测细胞活力以评估损伤程度（蓝色下方面板）。(B) 24小时血清、葡萄糖与氧剥夺的实验结果。(C) 36小时葡萄糖及氧剥夺后再灌注3小时的实验结果。*代表P值≤0.01，**代表P值≤0.001（共3次独立重复实验）。

图4：甲硫氨酸亚砜还原酶敲除细胞对氧化应激的响应。(A) 36小时缺血再灌注后的细胞活力（共3次独立重复实验）。(B) 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。

补充图1：甲硫氨酸亚砜还原酶敲除细胞系的构建。(A)、(C)、(E)与(G)展示了敲除甲硫氨酸亚砜还原酶家族各基因的策略，红色剪刀代表CRISPR编辑位点。(B)、(D)、(F)与(H)展示了以低分子量条带为特征的纯合敲除细胞系的PCR基因分型结果。(I) 的蛋白质印迹结果验证了CRISPR/Cas9介导的Msra-/-敲除细胞系中MSRA的敲除效果，以野生型小鼠肝脏、野生型AML12细胞与重组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。

补充图2：单个甲硫氨酸亚砜还原酶B家族敲除细胞系对氧化应激的响应。(A) 36小时缺血再灌注后的细胞活力（共3次独立重复实验）。(B) 野生型AML12细胞对百草枯的剂量响应实验结果（共3次独立重复实验）。(C) 经90 μM百草枯孵育2天后的细胞活力（共4次独立重复实验）。采用学生t检验确定显著性差异：*代表P值≤0.01，**代表P值≤0.001。