Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: SIRT3 blocks myofibroblast differentiation and pulmonary fibrosis by preventing mitochondrial DNA damage

doi: 10.1152/ajplung.00188.2016

Figure Lengend Snippet: SIRT3 controls myofibroblast differentiation independently of its ability to reduce ROS levels. A: human lung fibroblasts were transduced with Ad.SIRT3 or the control adenovirus and then stimulated with TGF-β1 for 48 h. In another group, TGF-β1-stimulated myofibroblasts were treated with the antioxidant EUK134 (10 µM). Cells were stained with CM-H2DCFDA and ROS levels were measured by fluorescence-activated cell sorter. B: quantification of mean fluorescence intensity in different groups of cells. Means ± SE, n = 4. C: cell lysates were analyzed by Western blotting with desired antibodies. D: quantification of relative protein levels in different groups of cells, means ± SE, n = 4. E: quantification of TGF-β1-mediated cell death. Means ± SE, n = 4. ns, Not significant.

Article Snippet: However, our studies showed that treatment of cells with an antioxidant, EUK134, which is a biomimetic of SOD2 and catalase, was unable to prevent TGF-β 1 -induced myofibroblast differentiation, even though it was capable of blocking cellular ROS levels.

Techniques: Transduction, Control, Staining, Fluorescence, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: SIRT3 blocks myofibroblast differentiation and pulmonary fibrosis by preventing mitochondrial DNA damage

doi: 10.1152/ajplung.00188.2016

Figure Lengend Snippet: SIRT3 controls OGG1 levels and protects cells from TGF-β1-induced mtDNA damage. Human lung fibroblasts were transduced with Ad.SIRT3 or the control adenoviruses or pretreated with the antioxidant EUK134 as indicated, and then stimulated with TGF-β1 for 48 h. A: relative SIRT3 levels in control and TGF-β1-treated cells as measured by Western blotting, means ± SE, n = 3. B: mtDNA damage was assessed by PCR amplification of DNA as described in materials and methods. Values are means ± SE, n = 4. C: cell lysate was subjected to Western blotting with antibodies against OGG1 and actin. D: quantification of relative OGG1 protein levels in different groups of cells, means ± SE, n = 3. E: quantification of OGG1 mRNA levels in control and TGF-β1-treated cells, means ± SE, n = 3. F: cell lysates were analyzed by Western blotting for the indicated proteins. Note that, while SIRT3 overexpression restores OGG1 levels, EUK134 treatments did not. G: OGG1 was depleted in human lung fibroblasts by using OGG1 siRNA. These cells were later transduced with Ad.SIRT3 or the control adenoviruses for 24 h followed by TGF-β1 treatment. SIRT3 augmentation failed to suppress α-SMA expression in absence of OGG1. ns, Not significant.

Article Snippet: However, our studies showed that treatment of cells with an antioxidant, EUK134, which is a biomimetic of SOD2 and catalase, was unable to prevent TGF-β 1 -induced myofibroblast differentiation, even though it was capable of blocking cellular ROS levels.

Techniques: Transduction, Control, Western Blot, Amplification, Over Expression, Expressing