Journal: Cell Death & Disease

Article Title: SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells

doi: 10.1038/s41419-023-06169-1

Figure Lengend Snippet: a , b Reanalysis of the database obtained from GEO database (GSE30122) on human kidney specimens from patients with DKD (healthy control n = 11, DKD n = 10) showed that SIRT2 was significantly downregulated, and FN1 and COL3A1 were significantly upregulated in the tubulointerstitial (Tublnt) from DKD patients. c Reanalysis of the data obtained from the Nephrin database showed that SIRT2 was significantly downregulated in the Tublnt from DKD patients (healthy control n = 3, DKD n = 11). d Reanalysis of the data obtained from the Nephrin database (Ju CKD Tublnt) showed that SIRT2 was significantly downregulated, and FN1 and COL3A1 were significantly upregulated in the tublnt of kidney tissues from CKD patients ( n = 170). e Spearman correlations were analyzed between SIRT2 transcription level and eGFR (ml/min/1.73 m 2 ) in HLD and patients with CKD (left) or DKD (right), respectively. f , g Spearman correlation analysis of SIRT2 and FN1 , SIRT2 and COL3A1 in renal Tublnt of DKD patients. h–j Representative images of Masson’s trichrome staining ( h ) and SIRT2 immumohistochemical staining ( i ) in the kidney sections from patients with minimal change disease [control (Ctrl), n = 1], and DKD ( n = 8) (scale bar = 100 μm). j Quantitative analysis of Masson’s trichrome and SIRT2 immunofluorescence staining in the indicated human renal biopsies ( f and g ), the collagen deposition (left) and SIRT2 positive area (right) quantified in the kidney sections in 3 fields per patients at ×100 magnification. k–m Representative images of SIRT2 immumohistochemical staining in mouse fibrotic kidneys induced by UUO ( k ) or uIRI ( l ), compared with kidneys from mice after sham surgery (scale bar = 100 μm). m Western blot analysis of SIRT2 and β-actin expression in mouse fibrotic kidneys induced by UUO or uIRI, compared with contralateral kidneys (control). The quantitative results are shown in the bottom panel. β-actin was used as the loading control ( n = 6). For all panels, data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by a one-way ANOVA with a Bonferroni correction test ( b , c , d , j , m ). Spearman’s correlation is also shown ( e , f , g ).

Article Snippet: The commercial siRNA Ctrl (Cat. No. sc-37007, Santa Cruz), siRNA SIRT2 (sc-40988, Santa Cruz), siRNA SMURF2 (Cat. No. sc-41675, Santa Cruz), and siRNA P/CAF (Cat. No. sc-36198, Santa Cruz) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

Techniques: Control, Staining, Immunofluorescence, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells

doi: 10.1038/s41419-023-06169-1

Figure Lengend Snippet: Tubule-specific Sirt2 knockout mice ( Sirt2 tKO ) were generated by crossbreeding Pax8 -Cre mice with floxed Sirt2 mice on a C57BL/6 background. a–c Representative images of Sirius red and Masson’s trichrome staining in kidney sections from WT and Sirt2 tKO mice at day 7 post-surgery. The collagen deposition of Sirius red staining ( a , the upper panel) and Masson’s trichrome staining ( a , the bottom panel) were quantified in the kidney sections in 3 fields per mouse at ×100 magnification ( b is for Sirius red staining, c is for Masson’s trichrome staining [ n = 6]). d–f Representative images of SIRT2 immunohistochemical staining and E-cadherin immunofluorescence staining are shown in panel d, and the mRNA level of α-SMA ( e ) and E-cadherin ( f ) were determined by qPCR in the kidney samples from mice ( n = 6). The boxed areas in the upper panels are enlarged in the lower panels. The polarized distribution of E-cadherin in the basolateral membrane of tubules is indicated by white arrows; re-distribution of E-cadherin to the apical membrane of tubules is represented by red arrows. g , h Western blot analysis of CTGF, FN1, COL3A1, E-cadherin, α-SMA, and β-actin in the kidney samples from WT and Sirt2 tKO mice at day 7 post-surgery along with quantitative results shown in the right panel. β-actin was used as the loading control ( n = 6). For all panels, data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA with a Bonferroni correction test.

Article Snippet: The commercial siRNA Ctrl (Cat. No. sc-37007, Santa Cruz), siRNA SIRT2 (sc-40988, Santa Cruz), siRNA SMURF2 (Cat. No. sc-41675, Santa Cruz), and siRNA P/CAF (Cat. No. sc-36198, Santa Cruz) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

Techniques: Knock-Out, Generated, Staining, Immunohistochemical staining, Immunofluorescence, Membrane, Western Blot, Control

Journal: Cell Death & Disease

Article Title: SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells

doi: 10.1038/s41419-023-06169-1

Figure Lengend Snippet: AAV-Ctrl or AAV- Ggt (gamma-glutamyltransferase 1)- Sirt2 was injected into bilateral kidneys of mice in situ at five independent points in situ. After 2-week transfection, the mice received UUO surgery, and the contralateral kidneys were used as control. a–d Western blot analysis of CTGF, FN1, COL1A1, COL3A1, E-cadherin, α-SMA, and β-actin in the kidneys from mice at day 10 post-surgery, with quantitative results shown in the right panel, and β-actin used as the loading control ( n = 6). e Representative images of Sirius red staining, α-SMA immunohistochemical staining, and E-cadherin immunofluorescence staining were shown in the kidney from mice at day 10 post-surgery. The boxed areas in the upper panels are enlarged in the lower panels. The polarized distribution of E-cadherin in the basolateral membrane of tubules was indicated by white arrows; re-distribution of E-cadherin to the apical membrane of tubules is indicated by red arrows. Key in ( b ) also applies to ( d ). For all panels, the data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA with a Bonferroni correction test.

Article Snippet: The commercial siRNA Ctrl (Cat. No. sc-37007, Santa Cruz), siRNA SIRT2 (sc-40988, Santa Cruz), siRNA SMURF2 (Cat. No. sc-41675, Santa Cruz), and siRNA P/CAF (Cat. No. sc-36198, Santa Cruz) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

Techniques: Injection, In Situ, Transfection, Control, Western Blot, Staining, Immunohistochemical staining, Immunofluorescence, Membrane

Journal: Cell Death & Disease

Article Title: SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells

doi: 10.1038/s41419-023-06169-1

Figure Lengend Snippet: a HK2 cells were transfected with Ad-null or Ad- SIRT2 for 24 h, and treated with or without 2 ng/ml TGF-β for indicated times. Western blot analysis (left) of SIRT2, phosphorylation of SMAD2 (p-SMAD2), SMAD2, and β-actin, and the quantitative results are shown in the right panel ( n = 3). b–d HK2 cells were transfected with Ad-null or Ad- Sirt2 for 24 h, and treated with 6 ng/ml TGF-β for 6 h. Representative images of SMAD2 immunofluorescence staining are shown in ( b ), and densitometry quantification of nuclear levels of SMAD2 are shown in ( c ). d Western blot analyses (left) of SMAD2 in the fractions extracted from HK2 cells, and the quantitative results are shown in the right panel ( n = 3). e 3TP-Lux luciferase activity assay in HEK293T cells after transfection of the 3TP-Lux plasmid, a renilla plasmid, and Ad-null or Ad- SIRT2 for 24 h, followed by the treatment with or without 2 ng/ml TGF-β for 16 h. Relative luciferase activity is presented as folds of that in the cells with transfection of Ad-null ( n = 5). f , g HK2 cells were transfected with Ad-null or Ad- SIRT2 for 24 h, and treated with or without 2 ng/ml TGF-β for 24 h. f The mRNA level of FN1, CTGF , and α-SMA as determined by qPCR ( n = 5). g Western blot analysis of FN1, CTGF, α-SMA, SIRT2, and β-actin in HK2 cells, with quantitative results shown in the right panel ( n = 3). h PTECs isolated from Sirt2 knockout ( Sirt2 −/− ) or wild type (WT) mice were treated with 2 or 4 ng/ml TGF-β for 6 h. Western blot analysis of SIRT2, SMAD2, p-SMAD2, and β-actin, and the quantitative results are shown in the right panel ( n = 3). i PTECs isolated from Sirt2 knockout ( Sirt2 −/− ) or wild type (WT) mice were treated with 2 or 4 ng/ml TGF-β for 24 h. Western blot analysis of FN1, CTGF, α-SMA, SIRT2, and β-actin in HK2 cells, with quantitative results shown in the right panel ( n = 3). The key in ( a ) also applies to ( d – g ); the key in ( h ) also applies to ( i ). For all panels, data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by a one-way ANOVA with a Bonferroni correction test.

Article Snippet: The commercial siRNA Ctrl (Cat. No. sc-37007, Santa Cruz), siRNA SIRT2 (sc-40988, Santa Cruz), siRNA SMURF2 (Cat. No. sc-41675, Santa Cruz), and siRNA P/CAF (Cat. No. sc-36198, Santa Cruz) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

Techniques: Transfection, Western Blot, Phospho-proteomics, Immunofluorescence, Staining, Luciferase, Activity Assay, Plasmid Preparation, Isolation, Knock-Out

Journal: Cell Death & Disease

Article Title: SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells

doi: 10.1038/s41419-023-06169-1

Figure Lengend Snippet: a , b HK2 cell lysates were subjected to co-immunoprecipitation (Co-IP) with anti-SIRT2 or anti-SMAD2 antibody, respectively. Normal rabbit IgG was used as the control, and western blotting was conducted using the indicated antibodies. c HK2 cells transfected with Ad-Null or Ad- SIRT2 for 24 h, and treated with or without 2 ng/ml TGF-β for 6 h. HK2 cell lysates were subjected to Co-IP with anti-SMAD2 antibody, followed by western blotting using the indicated antibodies. The quantitative results are shown in the right panel ( n = 3). d HK2 cells transfected with Ad-Null or Ad- SIRT2 for 24 h, and treated with or without protease inhibitor MG132 (20 μM) in the present of TGF-β for 8 h. Western blot analysis of SIRT2, SMAD2, and β-actin in HK2 cells, with the quantitative results shown in the right panel ( n = 3). e HK2 cells were transfected with Ad-Null or Ad- SIRT2 , and treated with CHX under TGF-β stimulation for indicated times. Western blot analysis of SIRT2, SMAD2, and β-actin in HK2 cells, with the quantitative results shown in the right panel ( n = 3). f , g PTECs isolated from Sirt2 −/− mice and were transfected with Ad- Sirt2 , Ad- Sirt2 -N168A, and Ad- Sirt2 -H187Y for 24 h, followed by 6 h TGF-β stimulation. PTECs cell lysates subjected to Co-IP with anti-SMAD2 antibody, followed by western blotting using indicated antibodies, and the quantitative results were shown in the right panel ( n = 3). h , i SIRT2 siRNA (si SIRT2 ) or Ctrl siRNA transfected with HK2 cells for 24 h, followed by the treatment with/without 2 ng/ml TGF-β for 6 h. HK2 cell lysates were subjected to Co-IP with anti-SMAD2 antibody, followed by western blotting using indicated antibodies, and the quantitative results were shown in the bottom panel ( n = 3). j HK2 cells transfected with Ctrl siRNA or si SIRT2 for 24 h, followed by treated with or without protease inhibitor MG132 (20 μM) in the present of TGF-β for 8 h. Western blot analysis of SIRT2, SMAD2, and β-actin in HK2 cells, and the quantitative results were shown in the right panel ( n = 3). The key in ( a ) also applies to ( b – f ); the key in ( g ) also applies to ( h ). For all panels, data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by a one-way ANOVA with a Bonferroni correction test.

Article Snippet: The commercial siRNA Ctrl (Cat. No. sc-37007, Santa Cruz), siRNA SIRT2 (sc-40988, Santa Cruz), siRNA SMURF2 (Cat. No. sc-41675, Santa Cruz), and siRNA P/CAF (Cat. No. sc-36198, Santa Cruz) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Western Blot, Transfection, Protease Inhibitor, Isolation

Journal: Cell Death & Disease

Article Title: SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells

doi: 10.1038/s41419-023-06169-1

Figure Lengend Snippet: a SIRT2-SMAD2 K451 docking with the HDOCK server. High magnification of boxed areas is presented on the right. Left arrow indicates the SMAD2 protein 447-456 peptide; right arrow indicates SMAD2 protein K451 site. b The conservation of SMAD2 lysine 451 in different species. c , d HEK293T cells transfected with Ad-WT SMAD2 , Ad-mutant SMAD2 , or Ad -SIRT2 for 24 h, followed by the treatment of 2 ng/ml TGF-β for 6 h. HEK293T cell lysates were subjected to Co-IP with anti-SMAD2 antibody, followed by western blotting using indicated antibodies, and the quantitative results are shown in the right panel ( n = 3). d HEK293T cells were transfected with Ad-WT SMAD2 or Ad-mutant SMAD2 for 24 h, followed by the treatment of with or without 2 ng/ml TGF-β for 6 h. Western blot analyses of p-SMAD2 (Ser465), SMAD2, and β-actin in the whole cells lysates or fractions extracted from HEK293T cells, with quantitative results shown in the right panel ( n = 3). e HEK293T cells transfected with Ad-WT SMAD2 , Ad-mutant SMAD2 for 24 h, followed by the treatment with or without 2 ng/ml TGF-β for 2 h. Representative images of SIRT2 immunofluorescence staining are shown in the HEK293T cells (scale bar = 50 μm). f , g HEK239T cells were transfected with Ad-WT SMAD2 , Ad-mutant SMAD2 , or Ad- SIRT2 as indicated for 24 h, followed by the treatment with 2 ng/ml TGF-β for 6 h. g Western blot analyses of p-SMAD2 (Ser465), SMAD2, and β-actin in the whole cells lysates or fractions extracted from HEK293T cells, and the quantitative results are shown in the right panel ( n = 3). h Representative images of SIRT2 immunofluorescence staining are shown in the HEK293T cells (scale bar = 50 μm). Key in ( c ) also applies to ( d – g ). For all panels, data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA with a Bonferroni correction test.

Article Snippet: The commercial siRNA Ctrl (Cat. No. sc-37007, Santa Cruz), siRNA SIRT2 (sc-40988, Santa Cruz), siRNA SMURF2 (Cat. No. sc-41675, Santa Cruz), and siRNA P/CAF (Cat. No. sc-36198, Santa Cruz) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

Techniques: Transfection, Mutagenesis, Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence, Staining

Journal: Cell Death & Disease

Article Title: SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells

doi: 10.1038/s41419-023-06169-1

Figure Lengend Snippet: a , b HK2 cells transfected with Ad- SIRT2 and/or si SMURF2 for 24 h (Ad-Null and siCtrl were used as controls, respectively), and treated with 2 ng/ml TGF-β for 6 h. HK2 cell lysates were subjected to Co-IP with anti-SMAD2 antibody, followed by western blotting using indicated antibodies ( a ), with the quantitative results shown in b ( n = 3). c , d HK2 cells were transfected with Ad-Null or Ad- SIRT2 for 24 h, and treated with or without 2 ng/ml TGF-β for 6 h. HK2 cell lysates subjected to Co-IP with anti-SMAD2 antibody, followed by western blotting using indicated antibodies ( c ), with quantitative results shown in the ( d ) ( n = 3). e , f HEK293T cells were transfected with Ad-WT SMAD2 , Ad-mutant SMAD2 , or Ad- SIRT2 as indicated for 24 h, and treated with 2 ng/ml TGF-β for 6 h. HEK293T cell lysates subjected to Co-IP with anti-SMAD2 antibody, followed by western blotting using indicated antibodies ( f ). Quantitative results are shown in the right panel ( n = 3). g SMURF2-SMAD2 K451 docking with the HDOCK server. High magnification of boxed areas is presented on the left. Arrow indicates SMAD2 protein K451 site. h CBP-SMAD2 K451 docking with the HDOCK server. High magnification of boxed areas is presented on the right. Arrow indicates SMAD2 protein K451 site. i HEK239T cells were transfected with lentivirus- CBP or lentivirus empty vector for 48 h, and subjected to Ad-WT- SMAD2 or Ad-Mut- SMAD2 transfection as indicated for 24 h. HEK293T cell lysates subjected to Co-IP with anti-SMAD2 antibody, and western blotting using indicated antibodies. Quantitative results are shown in the right panel ( n = 3). j P/CAF-SMAD2 K451 docking with the HDOCK server. High magnification of boxed areas is presented on the left. Arrow indicates SMAD2 protein K451 site. k HEK239T cells were transfected with Ad-WT- SMAD2 , Ad-Mut- SMAD2 , or Ad- P/CAF as indicated for 24 h. HEK293T cell lysates subjected to Co-IP with anti-SMAD2 antibody, and western blotting using indicated antibodies. Quantitative results were shown in the right panel ( n = 3). Key in ( b ) also applies to ( d , g ). Key in ( i ) also applies to ( k ). For all panels, data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by a one-way ANOVA with a Bonferroni correction test.

Article Snippet: The commercial siRNA Ctrl (Cat. No. sc-37007, Santa Cruz), siRNA SIRT2 (sc-40988, Santa Cruz), siRNA SMURF2 (Cat. No. sc-41675, Santa Cruz), and siRNA P/CAF (Cat. No. sc-36198, Santa Cruz) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Mutagenesis, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells

doi: 10.1038/s41419-023-06169-1

Figure Lengend Snippet: a HK2 cells were treated with 2 ng/ml TGF-β for indicated times. Western blot analysis of SIRT2, SMAD3, p-SMAD3, and β-actin, and the quantitative results are shown in the right panel ( n = 3). b HK2 cells were transfected with Ad-null or Ad- Sirt2 for 24 h, and treated with 2 ng/ml TGF-β for 2 h. Western blot analyses (left) of SMAD3 in the fractions extracted from HK2 cells, and the quantitative results are shown in the right panel ( n = 3). c HK2 cell lysates were subjected to Co-IP with anti-SIRT2 or anti-SMAD3 antibody, respectively. Normal rabbit IgG was used as the control, and western blotting was conducted using indicated antibodies. d , e SIRT2-SMAD2 K341 and K378 docking with the HDOCK server. High magnification of boxed areas is presented on the left. Arrow indicates SMAD3 protein K341 and K378 site. f The conservation of SMAD3 lysine 341 and 378 in different species. g HEK293T cells were transfected with Ad-WT SMAD3 , Ad-mutant SMAD3 , or Ad -SIRT2 for 24 h, followed by the treatment of 2 ng/ml TGF-β for 6 h. HEK293T cell lysates were subjected to Co-IP with anti-acetylation or anti-SMAD3 antibody, followed by western blotting using indicated antibodies, and the quantitative results are shown in the right panel ( n = 3). For all panels, data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by a one-way ANOVA with a Bonferroni correction test.

Article Snippet: The commercial siRNA Ctrl (Cat. No. sc-37007, Santa Cruz), siRNA SIRT2 (sc-40988, Santa Cruz), siRNA SMURF2 (Cat. No. sc-41675, Santa Cruz), and siRNA P/CAF (Cat. No. sc-36198, Santa Cruz) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

Techniques: Western Blot, Transfection, Co-Immunoprecipitation Assay, Control, Mutagenesis

Journal: Cell Death & Disease

Article Title: SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells

doi: 10.1038/s41419-023-06169-1

Figure Lengend Snippet: a , b Kidney lysates subjected to Co-IP with anti-SMAD2 or anti-SMAD3 antibody in the WT and Sirt2 tKO mice at day 7 post-surgery, and western blotting using indicated antibodies ( a ). The quantitative results are shown in ( b and c ) ( n = 6). d , e Western blot analyses ( d ) of nuclear levels of TGF-β1, SMAD2, SMAD3 and H3 in the fractions extracted from the kidney of the WT and Sirt2 tKO mice at day 7 post-surgery. Quantitative results are shown in ( d ) ( n = 6). f Representative images of SMAD2 and SMAD3 immunofluorescence staining in the kidney of WT and Sirt2 tKO mice at day 7 post-surgery, and SMAD2 nuclear location quantified in the kidney sections in 3 fields per mouse at ×100 magnification ( n = 6). g qPCR analysis of the mRNA level of Col1a1 , Col1a2 , and Serpine1 in the kidney of mice ( n = 6). For all panels, data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by a one-way ANOVA with a Bonferroni correction test.

Article Snippet: The commercial siRNA Ctrl (Cat. No. sc-37007, Santa Cruz), siRNA SIRT2 (sc-40988, Santa Cruz), siRNA SMURF2 (Cat. No. sc-41675, Santa Cruz), and siRNA P/CAF (Cat. No. sc-36198, Santa Cruz) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence, Staining

Journal: Cell Death & Disease

Article Title: SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells

doi: 10.1038/s41419-023-06169-1

Figure Lengend Snippet: AAV-Ctrl or AAV- Ggt (gamma-glutamyltransferase 1)- shSmad2/3 was injected into bilateral kidneys of mice in situ at three independent points. After 2-week transfection, mice received UUO surgery, and the contralateral kidneys were used as control. a Western blot analysis of SMAD2, SMAD3, SIRT2 and β-actin in the kidneys from mice at day 10 post-surgery, with quantitative results shown in the right panel, and β-actin used as the loading control ( n = 6). b Representative images of SIRT2 immunohistochemical staining in the kidney sections. c–e Representative images of Sirius red and Masson’s trichrome staining were shown ( c ), and the collagen deposition ( c for Sirius red staining and ( d ) for Masson’s trichrome staining) was quantified in the kidney sections in 3 fields per mice at ×100 magnification ( n = 6). f Representative images of E-cadherin immunofluorescence staining were shown in the kidney from mice at day 10 post-surgery. g The mRNA level of Col1a1 , α-SMA, Col1a2 , and Serpine1 ( n = 6). h Western blot analysis of COL3A1, FN1, CTGF, and β-actin in the kidneys from mice at day 10 post-surgery, with quantitative results shown in the right panel, and β-actin used as the loading control ( n = 6). For all panels, data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA with a Bonferroni correction test.

Article Snippet: The commercial siRNA Ctrl (Cat. No. sc-37007, Santa Cruz), siRNA SIRT2 (sc-40988, Santa Cruz), siRNA SMURF2 (Cat. No. sc-41675, Santa Cruz), and siRNA P/CAF (Cat. No. sc-36198, Santa Cruz) were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions.

Techniques: Injection, In Situ, Transfection, Control, Western Blot, Immunohistochemical staining, Staining, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: Sirtuin 2, a Mammalian Homolog of Yeast Silent Information Regulator-2 Longevity Regulator, Is an Oligodendroglial Protein That Decelerates Cell Differentiation through Deacetylating α-Tubulin

doi: 10.1523/JNEUROSCI.4181-06.2007

Figure Lengend Snippet: SIRT2, tubulin acetylation, CNP, and oligodendrocyte differentiation. A–I, Double-immunofluorescence photomicrographs showing close correlation of SIRT2 expression (A, D) with CNP (B) and levels of α-tubulin acetylation (E) in cultured OLPs. Levels of total α-tubulin (H) were, however, not closely associated with SIRT2 expression (G). Morphologies of the immunonegative cells could also be appreciated in the respective bottom panels of phase-contrast photomicrographs (C, F, I). Note the increased α-tubulin acetylation and emergence/increases of SIRT2 and CNP immunoreactivities in the prematurity-stage oligodendrocytes, compared with the earlier-stage cells that showed no SIRT2 or CNP signals, and low levels of α-tubulin acetylation. Tub, α-Tubulin; AcTub, acetylated α-tubulin. Scale bars, 20 μm. J–L, Three-dimensional bar charts showing the differentiation progression of the arborization of OLP in relation to the emergence/increase of cellular expression levels of SIRT2, CNP and acetylated α-tubulin. S, I, C, and HC, Simple, intermediate, complex, and highly complex cellular arborization, respectively. BL, −, +, ++, and +++ are used to designate the levels of expression (see Materials and Methods). “% of OLP cells” on the z-axis represents, in each of the four expression levels, the percentage of cells in each complexity category relative to the total.

Article Snippet: Three small interfering RNA (siRNA) duplexes against rat SIRT2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001008368","term_id":"56605811"}} NM_001008368 ) at nucleotides 518–540, 1390–1412, and 1927–1949 were commercially synthesized (1stBase, Singapore).

Techniques: Immunofluorescence, Expressing, Cell Culture

Journal: The Journal of Neuroscience

Article Title: Sirtuin 2, a Mammalian Homolog of Yeast Silent Information Regulator-2 Longevity Regulator, Is an Oligodendroglial Protein That Decelerates Cell Differentiation through Deacetylating α-Tubulin

doi: 10.1523/JNEUROSCI.4181-06.2007

Figure Lengend Snippet: Molecular features of rat SIRT2. A, Western blots, showing specificity tests of rabbit polyclonal anti-SIRT2 antibody. Labels above and below blots indicate the protein sample origins and antibodies used for immunoblotting detection (IB), respectively. GST-SIRT2c, Recombinant GST-SIRT2 C terminus (209–351) protein expressed in Escherichia coli. PS, Preimmunization serum. The double arrows indicate the SIRT2 doublet detected by rabbit anti-SIRT2. The arrowhead points to the position of GST-SIRT2c. B, OLN-93 cells were transiently transfected (Tf) with either pEGFP-C1 vector (lane 1) or pEGFP-Sirt2 (lanes 2–4). Forty-eight hours after transfection, lysates of the cells were subjected to immunoprecipitation (IP) using anti-EGFP or irrelevant IgG, followed by IB with antibodies indicated below. C, Multitissue Western blot probed with rabbit polyclonal anti-SIRT2 antibody. The bottom panel shows the pan-actin immunoreactivity as loading control. SkM, Skeleton muscle. D–I, Western blot analyses comparing the expression profiles of SIRT2 and CNP in postnatal day 0, 3, 7, 10, 14, 21, 28, or 60 rat cerebrum, cerebellum, and spinal cord, respectively. Each lane was loaded with 20 μg of soluble tissue lysate. Signals for β-actin served as loading controls. Quantitative analyses of expression levels of SIRT2 and CNP relative to β-actin in the different CNS regions are shown on the right (G–I). PD, Postnatal day.

Article Snippet: Three small interfering RNA (siRNA) duplexes against rat SIRT2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001008368","term_id":"56605811"}} NM_001008368 ) at nucleotides 518–540, 1390–1412, and 1927–1949 were commercially synthesized (1stBase, Singapore).

Techniques: Western Blot, Recombinant, Transfection, Plasmid Preparation, Immunoprecipitation, Control, Expressing

Journal: The Journal of Neuroscience

Article Title: Sirtuin 2, a Mammalian Homolog of Yeast Silent Information Regulator-2 Longevity Regulator, Is an Oligodendroglial Protein That Decelerates Cell Differentiation through Deacetylating α-Tubulin

doi: 10.1523/JNEUROSCI.4181-06.2007

Figure Lengend Snippet: Distribution of SIRT2 mRNA and protein in rat CNS. A–F, In situ hybridization histochemistry (A–D) and immunohistochemistry (E, F) results showing the distribution of SIRT2-expressing cells in the cerebellum (A, E), cerebral cortex (B, F), caudate–putamen (C), and cervical spinal cord (D). III, V, and VI, Layers III, V, and VI of cerebral cortex; cc, corpus callosum; Gr, granule cell layer of cerebellar cortex; Mol, molecular layer of cerebellar cortex; Pur, Purkinje cell layer of cerebellar cortex; GM, gray matter of spinal cord; WM, white matter of spinal cord. Scale bars: A–D, 50 μm; E, F, 20 μm.

Article Snippet: Three small interfering RNA (siRNA) duplexes against rat SIRT2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001008368","term_id":"56605811"}} NM_001008368 ) at nucleotides 518–540, 1390–1412, and 1927–1949 were commercially synthesized (1stBase, Singapore).

Techniques: In Situ Hybridization, Immunohistochemistry, Expressing

Journal: The Journal of Neuroscience

Article Title: Sirtuin 2, a Mammalian Homolog of Yeast Silent Information Regulator-2 Longevity Regulator, Is an Oligodendroglial Protein That Decelerates Cell Differentiation through Deacetylating α-Tubulin

doi: 10.1523/JNEUROSCI.4181-06.2007

Figure Lengend Snippet: SIRT2 is an oligodendroglial protein. A–H, Double-immunofluorescence labeling of SIRT2 (red in all panels) plus CNP (A–D), GFAP (E), OX42 (F), neurofilament (G, H), or MBP (I, J) in the rat cerebellar cortex (A, E), caudate–putamen (B, F), cerebral cortex (I; layers III and IV), dentate gyrus (J), cross (C, G) or longitudinal sections (D, H) of cervical spinal white (C, G) or gray (D, H) matter. The inset in A or E is the observation of the marked area at a higher magnification. In A–D, note that, although it overlapped CNP (green) in most oligodendrocytic processes, SIRT2 (red) was undetected or only faintly positive in most oligodendroglial cell bodies that showed strong CNP staining (open arrowheads). K, L, On longitudinal sections of cervical spinal white matter, double-immunofluorescence labeling (K) of SIRT2 (red) together with pan-sodium channel (NavP, green), or triple labeling (L) of SIRT2 (red) together with NavP (green) and potassium channel Kv1.2 (green) showing SIRT2 distribution in the juxtanodal and paranodal domains of myelin sheath. The arrows point to examples of the NavP clusters at the nodes of Ranvier. The inset in L shows an example from the marked area at a higher magnification. Note the SIRT2-positive bands flanking the node of Ranvier and in between the strongly Kv1.2-positive juxtaparanodes. Gr, Granule cell layer of cerebellar cortex (A, E) or the dentate gyrus (J). Mol, Molecular layer of cerebellar cortex; Pur, Purkinje cell layer of cerebellar cortex. Scale bars: B, D, F, H–J, 20 μm; A, E, 50 μm; C, G, K, L, 10 μm.

Article Snippet: Three small interfering RNA (siRNA) duplexes against rat SIRT2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001008368","term_id":"56605811"}} NM_001008368 ) at nucleotides 518–540, 1390–1412, and 1927–1949 were commercially synthesized (1stBase, Singapore).

Techniques: Immunofluorescence, Labeling, Staining

Journal: The Journal of Neuroscience

Article Title: Sirtuin 2, a Mammalian Homolog of Yeast Silent Information Regulator-2 Longevity Regulator, Is an Oligodendroglial Protein That Decelerates Cell Differentiation through Deacetylating α-Tubulin

doi: 10.1523/JNEUROSCI.4181-06.2007

Figure Lengend Snippet: SIRT2 localization in oligodendrocytes/myelin sheaths under electron microscope. A, Immunoelectron micrograph of cervical spinal cord showing occasional SIRT2 immunoreactivity in the perikaryal cytoplasm and in marginal heterochromatin clumps of nucleus of oligodendrocyte (denoted by an asterisk). B–F, Immunoelectron micrographs of cervical spinal cord (B, C, F) or corpus callosum (D, E) showing SIRT2 immunoreactivity in the cytoplasm-containing outer layer of myelin sheaths (D, arrow) or the juxtanodal loops of myelin sheaths (B, C, E, F, open arrowheads) on the sides of the nodes of Ranvier (marked by “n” in the middle of the axons). Scale bars: A–D, F, 0.5 μm; E, 0.2 μm.

Article Snippet: Three small interfering RNA (siRNA) duplexes against rat SIRT2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001008368","term_id":"56605811"}} NM_001008368 ) at nucleotides 518–540, 1390–1412, and 1927–1949 were commercially synthesized (1stBase, Singapore).

Techniques: Microscopy

Journal: The Journal of Neuroscience

Article Title: Sirtuin 2, a Mammalian Homolog of Yeast Silent Information Regulator-2 Longevity Regulator, Is an Oligodendroglial Protein That Decelerates Cell Differentiation through Deacetylating α-Tubulin

doi: 10.1523/JNEUROSCI.4181-06.2007

Figure Lengend Snippet: Cytoplasmic α-tubulin is the substrate of SIRT2 in OLN93 cells. A, OLN-93 cells were transiently transfected (Tf) as indicated, lysed, and immunoprecipitated with anti-EGFP. The immunoprecipitates were then added to OLN-93 cell lysate (as the source for acetylated α-tubulin) together with various combinations of NAD (1 mm) or NAM (5 mm), and incubated for 2 h at room temperature. Samples after that were subjected to Western blot analyses (5 μg each lane). B, C, OLN-93 cells were transiently transfected with pXJ-Sirt2, lysed, and centrifuged; the supernatant (B, C) and pellet (C) of the cell lysate were subjected to Western blots. Crtl, Normal OLN-93 cell lysate. The bar charts on the top of B represent the densitometric analysis of the bands in lanes 1–4. The band densities of acetylated-lysine in lane 2 and acetylated-α-tubulin in lane 4 (both of which transfected with pXJ-Sirt2) are expressed as percentages of the control groups in lanes 1 and 3, respectively.

Article Snippet: Three small interfering RNA (siRNA) duplexes against rat SIRT2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001008368","term_id":"56605811"}} NM_001008368 ) at nucleotides 518–540, 1390–1412, and 1927–1949 were commercially synthesized (1stBase, Singapore).

Techniques: Transfection, Immunoprecipitation, Incubation, Western Blot, Control

Journal: The Journal of Neuroscience

Article Title: Sirtuin 2, a Mammalian Homolog of Yeast Silent Information Regulator-2 Longevity Regulator, Is an Oligodendroglial Protein That Decelerates Cell Differentiation through Deacetylating α-Tubulin

doi: 10.1523/JNEUROSCI.4181-06.2007

Figure Lengend Snippet: Inhibitory effects of SIRT2 overexpression on oligodendrocyte differentiation. A–H, Sirt2-transfected OLP cells showed decreased α-tubulin acetylation levels (A, arrow), and simpler morphology compared with neighboring untransfected cells (A, C, F, G), with cells transfected with SIRT2H150Y, the partially inactive form of SIRT2 deacetylase (B), and with pEGFP-C1 or JN141-transfected controls (E, H, D). Total α-tubulin levels in the Sirt2-transfected cells were not significantly affected (C). For the visualization of the much higher intensity of overexpressed SIRT2 signals in the transfected cells, normal endogenous SIRT2 signals in the neighboring cells, if any, were underexposed and therefore could not be optimally appreciated in some of the panels (A, B, C, G). I, Statistical analyses of morphological maturation of primary OLPs transfected with pXJ-Sirt2, pXJ-Sirt2N131A, pXJ-Sirt2H150Y, and pXJ-JN141 plasmids. Data are from three independent experiments (n > 300 transfected cells for each transfection group). ***p < 0.001 (Student's t tests). Error bars indicate SD. J–O, pEGFP-Sirt2 transfected OLN93 cells showed much fewer cell processes (J), in comparison with cells transfected by pXJ-JN alone (K) or pXJ-JN plus pEGFP-C1 (M). When cotransfected with pXJ-JN, pEGFP-Sirt2 counterregulated the arborization-promoting effect of the former (L), whereas the inhibitory effect of pEGFP-Sirt2N131A was much less obvious (N). Addition of nicotinamide (5 mm) to the culture medium partly abolished the arborization-inhibiting effect of SIRT2 (O). Cells in all groups except those shown in O were transfected in the presence of NAD. P, Statistical analyses showing the counteractive effect of overexpressed SIRT2 on JN overexpression-promoted OLN-93 cellular arborization. OLN-93 cells were transfected or treated as indicated in J–O. Data are from two independent experiments (n > 300 transfected cells for each transfection group). Plasmids/drugs for transfection/treatment of cultures are indicated on the top right of A–H and J–O, and antibodies for immunofluorescence on the bottom left. Refer to Figure 6 for abbreviations. Scale bars, 20 μm.

Article Snippet: Three small interfering RNA (siRNA) duplexes against rat SIRT2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001008368","term_id":"56605811"}} NM_001008368 ) at nucleotides 518–540, 1390–1412, and 1927–1949 were commercially synthesized (1stBase, Singapore).

Techniques: Over Expression, Transfection, Histone Deacetylase Assay, Comparison, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: Sirtuin 2, a Mammalian Homolog of Yeast Silent Information Regulator-2 Longevity Regulator, Is an Oligodendroglial Protein That Decelerates Cell Differentiation through Deacetylating α-Tubulin

doi: 10.1523/JNEUROSCI.4181-06.2007

Figure Lengend Snippet: Prolonged SIRT2 knockdown promoted OLP differentiation. A–F, siRNA knockdown of endogenous SIRT2 for 3 d (days 2–5 into differentiation) did not significantly alter the morphological complexity of OLPs or distribution of acetylated α-tubulin (A), CNP (C), and MBPs (E) compared with cells transfected with JN or HDAC6 siRNAs (B, D, F). OLPs within 10 d into differentiation had no JN or HDAC6 immunoreactivity (data not shown). Thus, the latter two siRNAs served here as negative controls. G–J, siRNA knockdown of endogenous SIRT2 for 6 or 8 d (days 2–8 or 2–10 into differentiation) promoted the differentiation of OLPs as evidenced by increased morphological complexity (I) and upregulation of acetylated α-tubulin and MBP expression (J) in comparison with untransfected or JN/HDAC6 siRNA-treated controls. No difference was observed in the expression of CNP, total α-tubulin and β-actin between the SIRT2 knockdown and the control groups (J). Examples of cell morphology and CNP expression in the 8 d SIRT2 knockdown and untransfected control are shown in G and H, respectively. I, Error bars indicate SD. Grayscale pictures under the graph represent typical examples for the four morphological hierarchies. HC, Highly complex; NC, untransfected negative control; siJN, JN siRNA 3235–3257; siSIRT2a, Sirt2 siRNA 518–540; siSIRT2b, Sirt2 siRNA 1390–1412. ***p < 0.001; **p < 0.01, Student's t tests. Refer to Figures 6 and ​and77 for additional abbreviations. Plasmids/siRNAs for transfection are indicated on the top of the columns, and antibodies for immunofluorescence on the bottom left of individual panels. Scale bars, 20 μm.

Article Snippet: Three small interfering RNA (siRNA) duplexes against rat SIRT2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001008368","term_id":"56605811"}} NM_001008368 ) at nucleotides 518–540, 1390–1412, and 1927–1949 were commercially synthesized (1stBase, Singapore).

Techniques: Knockdown, Transfection, Expressing, Comparison, Control, Negative Control, Immunofluorescence

Journal: Frontiers in Molecular Neuroscience

Article Title: SIRT2, ERK and Nrf2 Mediate NAD + Treatment-Induced Increase in the Antioxidant Capacity of PC12 Cells Under Basal Conditions

doi: 10.3389/fnmol.2019.00108

Figure Lengend Snippet: The SIRT2 inhibitor AGK2 prevented the NAD + -induced increases in the glutathione levels, GSH/GSSG ratio, glutamylcysteine ligase (GCL) level and nuclear Nrf2 level of PC12 cells. (A) AGK2 prevented the NAD + -induced increase in the GSH levels (effect of NAD + treatment, F (1,36) = 15.91, p = 0.0003; effect of AGK2 treatment, F (1,36) = 4.282, p = 0.0457; effect of AGK2 * NAD + treatment, F (1,36) = 6.875, p = 0.0127). (B) AGK2 prevented the NAD + -induced increase in the GSSG levels (effect of NAD + treatment, F (1,36) = 25.84, p < 0.0001; effect of AGK2 treatment, F (1,36) = 2.402, p = 0.1299; effect of AGK2 * NAD + treatment, F (1,36) = 7.422, p = 0.0099). (C) AGK2 prevented the NAD + -induced increase in the GSH/GSSG ratio (effect of NAD + treatment, F (1,36) = 20.21, p < 0.0001; effect of AGK2 treatment, F (1,36) = 8.883, p = 0.0051; effect of AGK2 * NAD + treatment, F (1,36) = 5.841, p = 0.0209). (D) AGK2 prevented the NAD + -induced increase in the total glutathione level (effect of NAD + treatment, F (1,36) = 4.292, p = 0.0455; effect of AGK2 treatment, F (1,36) = 0.1002, p = 0.7534; effect of AGK2 * NAD + treatment, F (1,36) = 1.769, p = 0.1919). For (A–D) , the assays were conducted after the cells were treated with 1 mM NAD + and 5 μM AGK2 for 24 h. (E) AGK2 prevented the NAD + -induced increase in the GCLc mRNA level of the cells, assessed at 6 h after the NAD + treatment (effect of NAD + treatment, F (1,44) = 15.46, p = 0.0003; effect of AGK2 treatment, F (1,44) = 10.24, p = 0.0026; effect of AGK2 * NAD + treatment, F (1,44) = 15.59, p = 0.0003). (F) AGK2 prevented the NAD + -induced increase in the GCLm mRNA level of the cells, assessed at 6 h after the NAD + treatment (effect of NAD + treatment, F (1,44) = 52.53, p < 0.0001; effect of AGK2 treatment, F (1,44) = 9.449, p = 0.0036; effect of AGK2 * NAD + treatment, F (1,44) = 20.06, p < 0.0001). (G) Representative western blot bands of GCLc and GCLm. (H) AGK2 prevented the NAD + -induced increases in the protein levels of GCLc in PC12 cells, assessed at 12 h after the NAD + treatment (effect of NAD + treatment, F (1,36) = 9.982, p = 0.0032; effect of AGK2 treatment, F (1,36) = 5.051, p = 0.0245; effect of AGK2 * NAD + treatment, F (1,36) = 3.86, p = 0.0572). (I) AGK2 prevented the NAD + -induced increases in the protein levels of GCLm in PC12 cells, assessed at 12 h after the NAD + treatment (effect of NAD + treatment, F (1,36) = 9.831, p = 0.0034; effect of AGK2 treatment, F (1,36) = 2.646, p = 0.1125; effect of AGK2 * NAD + treatment, F (1,36) = 5.171, p = 0.029). (J) AGK2 prevented the NAD + -induced increase in the Nrf2 mRNA level of the cells, assessed at 6 h after the NAD + treatment (effect of NAD + treatment, F (1,36) = 9.982, p = 0.0032; effect of AGK2 treatment, F (1,36) = 5051, p = 0.0245; effect of AGK2 * NAD + treatment, F (1,36) = 3.86, p = 0.0572). (K) Representative western blot bands of nuclear Nrf2. (L) AGK2 prevented the NAD + -induced nuclear translocation of Nrf2, assessed at 1 h after the cells were treated with 1 mM NAD + and 5 μM AGK2 (effect of NAD + treatment, F (1,32) = 3.753, p = 0.0616; effect of AGK2 treatment, F (1,32) = 7.13, p = 0.0118; effect of AGK2 * NAD + treatment, F (1,32) = 10.28, p = 0.0030). The data were pooled from four independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: For each well, 600 μl serum-free media containing 33.33 nM of each of the three SIRT2 siRNA oligos and 4 μl lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) were added.

Techniques: Western Blot, Translocation Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: SIRT2, ERK and Nrf2 Mediate NAD + Treatment-Induced Increase in the Antioxidant Capacity of PC12 Cells Under Basal Conditions

doi: 10.3389/fnmol.2019.00108

Figure Lengend Snippet: SIRT2 silencing prevented NAD + -induced increases in the glutathione levels, GSH/GSSG ratio, GCL level, and Nrf2 level of PC12 cells. (A) SIRT2 siRNA decreased the protein levels of SIRT2 in PC12 cells. (B) SIRT2 silencing prevented the NAD + -induced increase in the GSH level of the cells (effect of NAD + treatment, F (1,44) = 105.2, p < 0.0001; effect of SIRT2 siRNA, F (1,44) = 16.01, p = 0.0002; effect of SIRT2 siRNA * NAD + treatment, F (1,44) = 50.4, p < 0.0001). (C) Effects of SIRT2 siRNA and NAD + on GSSG levels (effect of NAD + treatment, F (1,44) = 1.544, p = 0.2206; effect of SIRT2 siRNA, F (1,44) = 0.0108, p = 9177; effect of SIRT2 siRNA * NAD + treatment, F (1,44) = 3.279, p = 0.077). (D) SIRT2 silencing prevented the NAD + -induced increase in the GSH/GSSG ratio of the cells (effect of NAD + treatment, F (1,44) = 12.21, p = 0.0011; effect of SIRT2 siRNA, F (1,44) = 4.623, p = 0.0371; effect of SIRT2 siRNA * NAD + treatment, F (1,44) = 3.336, p = 0.0746). (E) SIRT2 silencing prevented the NAD + -induced increase in the total glutathione level of the cells. (effect of NAD + treatment, F (1,44) = 123.6, p < 0.0001; effect of SIRT2 siRNA, F (1,44) = 13.73, p = 0.0006; effect of SIRT2 siRNA * NAD + treatment, F (1,44) = 56.69, p < 0.0001). (F) SIRT2 silencing prevented the NAD + -induced increase in the GCLc mRNA level of the cells (effect of NAD + treatment, F (1,44) = 5.452, p = 0.0242; effect of SIRT2 siRNA, F (1,44) = 8.229, p = 0.0063; effect of SIRT2 siRNA * NAD + treatment, F (1,44) = 10.69, p = 0.0021). (G) SIRT2 silencing prevented the NAD + -induced increase in the GCLm mRNA level of the cells (effect of NAD + treatment, F (1,44) = 32.68, p < 0.0001; effect of SIRT2 siRNA, F (1,44) = 10.09, p = 0.0027; effect of SIRT2 siRNA * NAD + treatment, F (1,44) = 4.664, p = 0.0363). (H) SIRT2 silencing prevented the NAD + -induced increase in the Nrf2 mRNA level of the cells (effect of NAD + treatment, F (1,44) = 305, p < 0.0001; effect of SIRT2 siRNA, F (1,44) = 178.7, p < 0.0001; effect of SIRT2 siRNA * NAD + treatment, F (1,44) = 178.7, p < 0.0001). The cells were treated with SIRT2 siRNA for 6 h. After the media was replaced with Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, the cells were treated with NAD + . RT-PCR assays were conducted at 6 h after the NAD + treatment, while glutathione assays were conducted at 24 h after the NAD + treatment. The data were pooled from four independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: For each well, 600 μl serum-free media containing 33.33 nM of each of the three SIRT2 siRNA oligos and 4 μl lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) were added.

Techniques: Modification, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Molecular Neuroscience

Article Title: SIRT2, ERK and Nrf2 Mediate NAD + Treatment-Induced Increase in the Antioxidant Capacity of PC12 Cells Under Basal Conditions

doi: 10.3389/fnmol.2019.00108

Figure Lengend Snippet: Both the SIRT2 inhibitor AGK2 and SIRT2 silencing prevented the NAD + -induced increase in phosphorylated ERK in PC12 cells. (A) AGK2 prevented the NAD + -induced increase in phosphorylated ERK. The cells were treated with 5 μM AGK2 and 1 mM NAD + for 0.5 h (effect of NAD + treatment, F (1,32) = 15.89, p = 0.0004; effect of AGK2 treatment, F (1,32) = 2.462, p = 0.1265; effect of AGK2 * NAD + treatment, F (1,32) = 7.258, p = 0.0111). (B) SIRT2 silencing prevented the NAD + -induced increase in phosphorylated ERK. After the cells were treated with SIRT2 siRNA for 6 h, the media was replaced with DMEM containing 10% fetal bovine serum (effect of NAD + treatment, F (1,32) = 6.415, p = 0.0164; effect of SIRT2 siRNA, F (1,32) = 8.138, p = 0.0075; effect of SIRT2 siRNA * NAD + treatment, F (1,32) = 2.114, p = 0.1557). Then the cells were treated with 1 mM NAD + for 0.5 h. * p < 0.05; ** p < 0.01.

Article Snippet: For each well, 600 μl serum-free media containing 33.33 nM of each of the three SIRT2 siRNA oligos and 4 μl lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) were added.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: SIRT2, ERK and Nrf2 Mediate NAD + Treatment-Induced Increase in the Antioxidant Capacity of PC12 Cells Under Basal Conditions

doi: 10.3389/fnmol.2019.00108

Figure Lengend Snippet: Diagrammatic presentation of the mechanisms underlying the relationships among NAD + , ERK, SIRT2, Nrf2, GCL, glutathione metabolism, and antioxidant capacity of cells.

Article Snippet: For each well, 600 μl serum-free media containing 33.33 nM of each of the three SIRT2 siRNA oligos and 4 μl lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) were added.

Techniques:

Journal: Cell Death Discovery

Article Title: Inhibitor of DNA binding 2 (Id2) mediates microtubule polymerization in the brain by regulating αK40 acetylation of α-tubulin

doi: 10.1038/s41420-021-00652-4

Figure Lengend Snippet: A HEK293T cells were transfected as indicated and the cell lysates were subjected to GST pull-down and immunoblotting as indicated. B In vitro deacetylase assay was performed with GFP–Id2 and Flag–Sirt2 proteins. The bar graphs represent the quantification of the acetyl-α-tubulin to α-tubulin ratio. C , D GST–Id2 and GFP–HDAC6 ( C ) or GFP–αTAT ( D ) transfected cell lysates were subjected to immunoblotting as indicated. E Primary cultured neurons were co-transfected with GFP–Sirt2 or GFP vector control and RFP–Id2 or RFP vector on DIV 3 and fixed on DIV 5. Neurons were stained with anti-NeuN (blue) and anti-acetylated tubulin (purple). Quantification of fluorescence intensity and axon length is shown at the right. Scale bar, 20 μm. * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001. All values are mean ± SEM and the representative images shown are from at least three independent experiments.

Article Snippet: The siRNA for silencing Id2 (5′-GAGCUUAUGUCGAAUGAUAUU-3′) and the siRNA for silencing Sirt2 (5′-AATCTCCACATCCGCAGGCAT-3′) were obtained from Genolution (Republic of Korea).

Techniques: Transfection, Western Blot, In Vitro, Histone Deacetylase Assay, Cell Culture, Plasmid Preparation, Staining, Fluorescence

Journal: Cell Death Discovery

Article Title: Inhibitor of DNA binding 2 (Id2) mediates microtubule polymerization in the brain by regulating αK40 acetylation of α-tubulin

doi: 10.1038/s41420-021-00652-4

Figure Lengend Snippet: A , B GST–Id2 and mammalian GFP–Sirt2 fragments ( A ) transfected cells were subjected to GST pull-down assay and immunoblotting as indicated. C , D Flag–Sirt2 and mammalian GST–α-tubulin fragments ( C ) transfected cells were subjected to GST pull-down and immunoblotting as indicated. E , F HEK293T cells were transfected with 0, 1, 3 μg of GFP–Id2 ( E ) or Flag–Sirt2 ( F ) with GST–α-tubulin, and lysates were subjected to GST pull-down and immunoblotting. The bar graphs represent the quantification of Sirt2 or Id2–α-tubulin binding. G , H Primary hippocampal neurons were transfected as indicated and the cell lysates were subjected to GST pull-down assay. I Proximity ligation assay (PLA) was conducted with scr, si-Id2, or si-Sirt2 expressing cells. Confocal images were shown tubulin-Id2 PLA staining (left) and tubulin-Sirt2 PLA staining (right). Nuclei were stained by DAPI (blue). Scale bar 5 μm. Quantification of tubulin-Id2 or tubulin-Sirt2 PLA puncta is shown as a bar graph. ** p < 0.005, *** p < 0.0005, **** p < 0.0001. All values are mean ± SEM and the representative images shown are from at least three independent experiments.

Article Snippet: The siRNA for silencing Id2 (5′-GAGCUUAUGUCGAAUGAUAUU-3′) and the siRNA for silencing Sirt2 (5′-AATCTCCACATCCGCAGGCAT-3′) were obtained from Genolution (Republic of Korea).

Techniques: Transfection, Pull Down Assay, Western Blot, Binding Assay, Proximity Ligation Assay, Expressing, Staining

Journal: Cell Death Discovery

Article Title: Inhibitor of DNA binding 2 (Id2) mediates microtubule polymerization in the brain by regulating αK40 acetylation of α-tubulin

doi: 10.1038/s41420-021-00652-4

Figure Lengend Snippet: A , B Protein levels in postmortem brain samples from AD patients and age-matched control (each n = 3) ( A ) and 5X-FAD and age-matched WT mice (each n = 3) ( B ) were determined by immunoblotting against anti-Id2, Akt1, Sirt2, and Tubulin antibodies. C , D The paraffin-embedded sections from the hippocampus of WT or 5X-FAD mice were immunostained with anti-NEUN (red) or anti-Id2 (green) ( C ) and with anti-Sirt2 (red) or anti-acetyl-α-tubulin (green) ( D ) antibodies, and DAPI (blue), respectively. Quantification of immunoreactivity for Id2 ( C ) or acetyl-α-tubulin and Sirt2 ( D ) is shown right (mean ± SEM of 12–18 sections from n = 4 mice per group). Scale bar 20 μm ( C ) and 10 μm ( D ). E Hippocampal neurons from 5X-FAD or WT mice were transfected with RFP-Id2-WT or S14A at DIV 14. Immunostaining was performed with anti-acetyl-α-tubulin (green) and Tubulin (blue). Scale bar 20 μm. Quantification of fluorescence intensity and axon length from more than three independent experiments is shown at the right. F , G 5X-FAD brain (P20) slice cultures were performed and maintained for 28 days. The slices were infected with AAV2-Id2 WT, S14A, or control at DIV 7 and maintained for an additional 20 days. Immunostaining was conducted with anti-acetyl-α-tubulin (red) and anti-NEUN (blue). Scale bar 5 μm. * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001 versus indicated. All values are mean ± SEM and the representative images shown are from at least three independent experiments.

Article Snippet: The siRNA for silencing Id2 (5′-GAGCUUAUGUCGAAUGAUAUU-3′) and the siRNA for silencing Sirt2 (5′-AATCTCCACATCCGCAGGCAT-3′) were obtained from Genolution (Republic of Korea).

Techniques: Western Blot, Transfection, Immunostaining, Fluorescence, Infection