Figure 2. ALCAT1 causes oxidative stress in C2C12 cells, and is up-regulated by ROS and by DIO.

The C2C12 cells stably overexpressing ALCAT1 or vector used in Fig 1 were analyzed for A, intracellular level of ROS before (control) and after treatment with H₂O₂; B, mtDNA copy number in response to H₂O₂ treatment; C, the expression of genes encoding antioxidant enzymes, including peroxiredoxin (Prdx), peroxiredoxin-6 related sequence (Prdx6-rs1), glutathione peroxidases (Gpx), superoxide dismutases (Sod), glutathione reductase (Gsr), nucleoredoxin (Nxn), sulfiredoxin 1 homolog (Srxn1), and thioredoxin reductase (Txnrd); and D, expression of genes encoding oxidant enzymes, including NADPH oxidase-4 (Nox4), dual oxidase-1 (Duox1), NAD(P)H quinone oxidoreductase-1 (Nqo1), and uncoupling protein-3 (Ucp3) by using an oxidative stress pathway real-time PCR microarray kit. E, ALCAT1 mRNA expression was up-regulated in isolated mouse cardiomyocytes in response to treatment with tert-butylhydroperoxide (T-Bu), an oxidant, and H₂O₂, as measured by RT-PCR analysis using β-actin as an internal control. F, ALCAT1 mRNA expression was analyzed in liver, heart, and skeletal muscle of ALCAT1^−/− mice (KO) and the wild type control (WT) mice fed a normal diet (ND) or high-fat diet (HFD) by RT-PCR analysis using β-actin as internal control, and quantified (G). SEM, *P<0.05, **P<0.01, compared to vector controls. N=3. *P < 0.05, ** P < 0.01.