DOI: 10.25444/nhlbi.23415557.v1
title: <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 & Medicine

description: This is a set of data, published in the journal of Free Radical Biology & 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.