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. 2014 Nov 25;26(8):1939–1959. doi: 10.1681/ASN.2014030289

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Copyright © 2015 by the American Society of Nephrology

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Figure 5. — SIRT1 activity was necessary for actin cytoskeleton maintenance in podocytes. (A) Oxidative stress detection by immunohistochemistry for nitrotyrosine in glomeruli of control or GN-induced mice. In NTS-injected mice, podocytes were positive for nitrotyrosine, indicating that oxidative stress was induced in GN induced by NTS. Scale bar, 50 μm. (B) WB analysis and densitometry for acetyl-histone H3 (Ac-HisH3) in cultured podocytes. EX-527, an SIRT1 inhibitor, significantly increased acetylation level of histone H3 in a dose-dependent manner in podocytes. (C) Detection of actin fiber by fluorescein–phalloidin staining in cultured podocytes. Lower panels are enlargements. Cultured podocytes were treated with SIRT1 inhibitors (EX-527, 100 µM; cambinol, 50 µM; or NAM, 10 mM) or vehicle (ethanol) for 24 hours and then treated with or without H₂O₂ (300 µM) for 24 hours. H₂O₂ was used to induce podocyte injury to mimic that in GN induced by NTS. Actin cytoskeleton derangement was markedly deteriorated in podocytes treated with both EX-527 and H₂O₂ compared with the cells treated with either EX-527 or H₂O₂. Similar results were confirmed using the other SIRT1 inhibitors cambinol or NAM. Scale bars, 50 μm. (D and E) Quantitative analysis of actin cytoskeleton derangement in podocytes treated with EX-527 (EX; 100 µM) and H₂O₂ (300 µM). (D) Mean score of actin cytoskeleton derangement and (E) ratio of the cells with severe derangement (corresponds to score=4) were measured. The derangement was significantly exacerbated by SIRT1 inhibition under oxidative stress conditions. (F) Fluorescein–phalloidin staining of cultured podocytes treated with SIRT1 activator under oxidative stress conditions. Lower panels are enlargements. Cultured podocytes were pretreated with SIRT1 activator resveratrol (200 µM) or vehicle (ethanol) for 3 hours and then cultured with high-dose H₂O₂ (high H₂O₂; 700 μM) for 1 hour. Resveratrol prevented actin cytoskeleton derangement induced by oxidative stress. Scale bars, 50 μm. (G and H) Quantitative analysis of actin cytoskeleton derangement in podocytes treated with resveratrol and H₂O₂. (G) Mean score of actin cytoskeleton derangement and (H) ratio of the cells with severe derangement were measured. The derangement was significantly attenuated by SIRT1 activation under oxidative stress conditions. RSV, resveratrol; vehi, vehicle (ethanol). *P<0.05 versus vehicle; **P<0.01 versus vehicle; ***P<0.001.

Figure 5. — SIRT1 activity was necessary for actin cytoskeleton maintenance in podocytes. (A) Oxidative stress detection by immunohistochemistry for nitrotyrosine in glomeruli of control or GN-induced mice. In NTS-injected mice, podocytes were positive for nitrotyrosine, indicating that oxidative stress was induced in GN induced by NTS. Scale bar, 50 μm. (B) WB analysis and densitometry for acetyl-histone H3 (Ac-HisH3) in cultured podocytes. EX-527, an SIRT1 inhibitor, significantly increased acetylation level of histone H3 in a dose-dependent manner in podocytes. (C) Detection of actin fiber by fluorescein–phalloidin staining in cultured podocytes. Lower panels are enlargements. Cultured podocytes were treated with SIRT1 inhibitors (EX-527, 100 µM; cambinol, 50 µM; or NAM, 10 mM) or vehicle (ethanol) for 24 hours and then treated with or without H₂O₂ (300 µM) for 24 hours. H₂O₂ was used to induce podocyte injury to mimic that in GN induced by NTS. Actin cytoskeleton derangement was markedly deteriorated in podocytes treated with both EX-527 and H₂O₂ compared with the cells treated with either EX-527 or H₂O₂. Similar results were confirmed using the other SIRT1 inhibitors cambinol or NAM. Scale bars, 50 μm. (D and E) Quantitative analysis of actin cytoskeleton derangement in podocytes treated with EX-527 (EX; 100 µM) and H₂O₂ (300 µM). (D) Mean score of actin cytoskeleton derangement and (E) ratio of the cells with severe derangement (corresponds to score=4) were measured. The derangement was significantly exacerbated by SIRT1 inhibition under oxidative stress conditions. (F) Fluorescein–phalloidin staining of cultured podocytes treated with SIRT1 activator under oxidative stress conditions. Lower panels are enlargements. Cultured podocytes were pretreated with SIRT1 activator resveratrol (200 µM) or vehicle (ethanol) for 3 hours and then cultured with high-dose H₂O₂ (high H₂O₂; 700 μM) for 1 hour. Resveratrol prevented actin cytoskeleton derangement induced by oxidative stress. Scale bars, 50 μm. (G and H) Quantitative analysis of actin cytoskeleton derangement in podocytes treated with resveratrol and H₂O₂. (G) Mean score of actin cytoskeleton derangement and (H) ratio of the cells with severe derangement were measured. The derangement was significantly attenuated by SIRT1 activation under oxidative stress conditions. RSV, resveratrol; vehi, vehicle (ethanol). *P<0.05 versus vehicle; **P<0.01 versus vehicle; ***P<0.001.