Journal: The Journal of Biological Chemistry

Article Title: SIRT2 deacetylase represses NFAT transcription factor to maintain cardiac homeostasis

doi: 10.1074/jbc.RA117.000915

Figure Lengend Snippet: Pathological hypertrophy reduces SIRT2 levels in heart. A, representative confocal images of vehicle or PE (20 μm for 48 h) treated neonatal primary cardiomyocytes stained with α-actinin and SIRT2. Scale bar = 20 μm. B, Western blot analysis of vehicle- or PE- or ISO- treated (20 μm for 48 h) primary cardiomyocytes probed for SIRT2. C, quantification of SIRT2 from B. Error bars, mean ± S.D.; *, p < 0.05. D, scatter plot representing HW/TL ratio of vehicle- or ISO-treated (10 mg/kg/day for 7 days) mice. n = 9 mice; error bars, mean ± S.D.; *, p < 0.05. E, scatter plot representing fractional shortening of vehicle- or ISO-treated (10 mg/kg/day for 7 days) mice. n = 6 mice; error bars, mean ± S.D.; *, p < 0.05. F, qPCR analysis of SIRT2, ANP, BNP, and PPIA in vehicle- or ISO-treated mice hearts. Cyclophilin A (PPIA) was used as negative control. n = 4 mice; error bars, mean ± S.D.; *, p < 0.05. G, Western blot analysis of vehicle- and/or ISO-treated mice heart samples for SIRT2 and ANP. n = 4 mice. H, quantification of SIRT2 from G. I, Western blot analysis of vehicle- or ISO-treated mice for SIRT2, n = 4 mice. J, quantification of SIRT2 from I.

Article Snippet: The following antibodies were used: α-actinin (Sigma-Aldrich), SIRT2 (EMD Millipore, Sigma-Aldrich, Cell Signaling Technology#), ANP (Abcam, Sunny Corp*), NFATc2 (Thermo Scientific), calcineurin (Thermo Scientific), NFAT (Thermo Scientific), acetyl lysine (EMD Millipore, Cell Signaling Technology), puromycin (Developmental Studies Hybridoma Bank), α-SMA (EMD Millipore), Fn1 (Santa Cruz Biotechnology), Col1a (Sigma-Aldrich), Col3a (Santa Cruz Biotechnology).

Techniques: Staining, Western Blot, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: SIRT2 deacetylase represses NFAT transcription factor to maintain cardiac homeostasis

doi: 10.1074/jbc.RA117.000915

Figure Lengend Snippet: SIRT2 depletion induces hypertrophy in cardiomyocytes. A, Western blot analysis confirming depletion of SIRT2 in cardiomyocytes transfected with SIRT2-specific siRNA. B, [3H]leucine incorporation into cellular proteins of neonatal rat cardiomyocytes transfected with control or SIRT2 siRNA. n = 5 mice; error bars, mean ± S.D.; *, p < 0.05. c.p.m., counts per minute. C, Western blot analysis of control and SIRT2-knockdown (SIRT2-KD) cardiomyocytes treated with vehicle or PE (20 μm) or ISO (20 μm) for 48 h and probed for ANP and SIRT2. D, representative confocal images of control and SIRT2-KD cardiomyocytes treated with vehicle or PE (20 μm) or ISO (20 μm) for 48 h and further stained with ANP and α-actinin or myomesin. Scale bar = 20 μm. E, Western blot analysis showing overexpression of SIRT2 in cardiomyocytes. F, [3H]leucine incorporation into total cellular protein of neonatal rat cardiomyocytes infected with SIRT2 (Ad-SIRT2) or control adenovirus (Ad-GFP) and then treated with vehicle or PE (20 μm) or ISO (20 μm) for 48 h. n = 5; error bars, mean ± S.D.; *, p < 0.05. c.p.m., counts per minute. G, Western blot analysis of Ad-null or Ad-SIRT2 expressing cardiomyocytes treated with vehicle or PE (20 μm) or ISO (20 μm) for 48 h and harvested after puromycin treatment (1 μm), and probed for puromycin, ANP, and SIRT2. H, quantification of puromycin incorporation from G. I, representative confocal images of control and SIRT2 overexpressing cardiomyocytes treated with vehicle or PE (20 μm) or ISO (20 μm) for 48 h and stained with ANP and α-actinin or myomesin. Scale bar = 20 μm. J, graph showing quantification of relative cardiomyocyte size measured from I.

Article Snippet: The following antibodies were used: α-actinin (Sigma-Aldrich), SIRT2 (EMD Millipore, Sigma-Aldrich, Cell Signaling Technology#), ANP (Abcam, Sunny Corp*), NFATc2 (Thermo Scientific), calcineurin (Thermo Scientific), NFAT (Thermo Scientific), acetyl lysine (EMD Millipore, Cell Signaling Technology), puromycin (Developmental Studies Hybridoma Bank), α-SMA (EMD Millipore), Fn1 (Santa Cruz Biotechnology), Col1a (Sigma-Aldrich), Col3a (Santa Cruz Biotechnology).

Techniques: Western Blot, Transfection, Control, Knockdown, Staining, Over Expression, Infection, Expressing