Journal: Cells

Article Title: Estetrol Enhances Mitochondrial Bioenergetics and Neurite Outgrowth in Cellular Models of Alzheimer’s Disease

doi: 10.3390/cells15050452

Figure Lengend Snippet: Impact of estrogen receptor antagonists on the E2- and E4-mediated increase in ATP levels in Control, APP and P301L cells. Cells were first pre-treated with 0.1 μM MPP (ERα antagonist), 0.1 μM PHTPP (ERβ antagonist), 0.1 μM fulvestrant (ER antagonist), or 0.1 μM G-15 (GPER1 antagonist) for 1 h and then treated with vehicle alone (Veh = DMSO), E2 0.1 μM, and E4 1 μM for 48 h. Data represent the ATP levels as mean ±SEM of three independent experiments, expressed as a percentage of the vehicle (Veh) condition. In addition, the mean of each independent experiment (biological replicates) is presented as circles (for Control cells), squares (for APP cells), or diamonds (for P301L cells). Total number of technical replicates per condition: n = 11–36 ( A ); n = 11–36 ( B ); and n = 12–32 ( C ). All the data were normalized on the CellTracker Blue fluorescence intensity, which corresponds to the area of living cells. * p < 0.05, ** p < 0.01, two-way ANOVA versus Veh/DMSO. MPP: Methyl-piperidino-pyrazole, E2: estradiol, E4: estetrol.

Article Snippet: MPP (#HY-103454), PHTPP (#HY-103456), Fulvestrant (#HY-103636) and G-15 (#HY-103449) were obtained from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control, Fluorescence

Journal: Frontiers in Immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: The effect of acute ethanol-exposure on mouse bone marrow derived macrophages (BMDM). Phagocytosis in BMDM exposed to vehicle or ethanol ± LPS to study phagocytosis using pHrodo bioparticles and SIRT2 expression. (A) Representative images of intracellular pHrodo bioparticles in vehicle or Ethanol-exposed WT-BMDM ± LPS. (B) Fluorescence quantification of pHrodo bioparticles in WT-BMDM (n=4 repetitions/group; * p<0.05). (C) SIRT2 protein expression detected by western blot in Vehicle- or Ethanol-exposed BMDM cells ± LPS. (D) Western blot image quantification of SIRT2 protein blot in vehicle or ethanol-exposed BMDM cells ± LPS (n = 4 blots/group; * p < 0.05). (E) Representative images of pHrodo bioparticles in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. (F) Fluorescence quantification of pHrodo bioparticles in Ethanol-exposed WT-BMDM, and SIRT2KO-BMDM ± LPS. * p < 0.05. (G) Representative images of pHrodo bioparticles in Ethanol-exposed WT-BMDM, co-treated with SIRT2 inhibitor AK-7 or DMSO ± LPS. (H) Fluorescence quantification of pHrodo bioparticles in Ethanol-exposed WT-BMDM with AK-7/DMSO ± LPS stimulation. * p < 0.05.

Article Snippet: PFKP ( Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc-393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Anti-rabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PU-N), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: Derivative Assay, Expressing, Fluorescence, Western Blot

Journal: Frontiers in Immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: The effect of acute ethanol-exposure-induced SIRT2 on PFKP expression in macrophages. (A) PFKP expression in WT-BMDM exposed to vehicle or ethanol ± LPS by western blot analysis. (B) PFKP western blot image quantification of PFKP protein in vehicle vs. ethanol exposed WT-BMDM, Y axis represents fold of vehicle-LPS (fold of vehicle control) (n = 4 blots; * p < 0.05). (C) PFKP expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. (D) Western blot image quantification of PFKP protein in ethanol exposed WT-BMDM and SIRT2KO-BMDM. Y axis represents fold of Ethanol-exposed WT-LPS (fold of WT- ethanol control) (n = 4 blots; * p < 0.05). (E) PFKP expression in Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. (F) Western blot image quantification of PFKP in AK-7 vs. DMSO treated-Ethanol-exposed WT-BMDM ± LPS, Y axis represents fold of Ethanol-exposed WT-LPS (fold of WT- ethanol control) (n = 4 blots; * p < 0.05).

Article Snippet: PFKP ( Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc-393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Anti-rabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PU-N), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: SIRT2-PFKP in vivo and in vitro interaction. (A) RAW264.7 cell macrophages (RAW) ± LPS. IP of whole-cell lysates using an anti-SIRT2 antibody followed by IB analysis of PFKP and SIRT2. IP with isotype IgG control antibody was used as a negative control. (B) Western blot analysis of PFKP and SIRT2 in the whole cell lysate used as input for the SIRT2 IP. (C) In-vitro interaction between SIRT2 and PFKP using SPR. SIRT2 protein immobilized onto sensor chip and the PFKP was flowed at various concentration (15.62, 31.25, 62.5, 125, 250, 500 and 1000nM). The response units on Y axis (RU) represent quantitative assessment of protein-protein interaction. (D) wtPFKP and control plasmid transfection and IP, using turbo-GFP-trap in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of acetyl lysine, turbo-GFP-wtPFKP and control for PFKP (turbo-GFP). HEK293T cell lysate without transfection used as a negative control. (E) Western blot analysis of control-PFKP (Turbo-GFP), wtPFKP (Turbo-GFP-wtPFKP), DDK-SIRT2 and CPA in whole cell lysate used as input for the turbo-GFP IP. (F) wtPFKP transfection and IP using magnetic-TUBEs in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of ubiquitination. (G) Western blot analysis of turbo-GFP wtPFKP, DDK-SIRT2, turbo-GFP and CPA in whole cell lysate used as an input for the TUBE IP.

Article Snippet: PFKP ( Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc-393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Anti-rabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PU-N), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: In Vivo, In Vitro, Control, Negative Control, Western Blot, Concentration Assay, Plasmid Preparation, Transfection, Ubiquitin Proteomics

Journal: Frontiers in Immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: Effect of K394R mutation on PFKP. (A, B) HEK293T cells transfected with wtPFKP/mtPFKP in the presence or absence of SIRT2. Western blot analysis of Turbo-GFP-wtPFKP, DDK-SIRT2, turbo-GFP (control for wtPFKP plasmid transfected) and CPA. (C) mtPFKP transfection and IP using turbo-GFP-trap in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of acetyl lysine, turbo-GFP-mtPFKP and turbo-GFP (control for wtPFKP plasmid transfected). Pulldown with HEK293T cell lysate without transfection was used as a negative control. (D) Western blot analysis of turbo-GFP mtPFKP, DDK-SIRT2, turbo-GFP and CPA in whole cell lysate, used as an input for the turbo-GFP IP. (E) mtPFKP transfection and IP using magnetic-TUBEs in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of ubiquitination. (F) Western blot analysis of turbo-GFP mtPFKP, DDK-SIRT2, turbo-GFP and CPA in whole cell lysate used as input for the TUBE IP.

Article Snippet: PFKP ( Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc-393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Anti-rabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PU-N), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: Mutagenesis, Transfection, Western Blot, Control, Plasmid Preparation, Negative Control, Ubiquitin Proteomics

Journal: Frontiers in Immunology

Article Title: SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure

doi: 10.3389/fimmu.2022.1079962

Figure Lengend Snippet: The effect of acute ethanol-exposure on SIRT2 expression and the effect of AK-7 on LC3-associated phagocytosis in Ethanol-exposed Human macrophages. Human macrophages were exposed to vehicle or ethanol ± LPS to study SIRT2 expression and LAP. SIRT2 expression was analyzed by immunostaining (A) Representative images of SIRT2 immunostaining in vehicle or Ethanol-exposed human macrophages ± LPS. (B) Fluorescence quantification of SIRT2 immunostaining in human macrophages (n=4; *p<0.05). (C) LAP in human macrophages with vehicle or ethanol-exposure ± LPS. Representative images of intracellular pHrodo bioparticles during phagocytosis and co-stained for LC3. (D) For each image, the co-localization of intracellular pHrodo (red) and LC3 (green) were determined as LAP and divided by the total numbers of nuclei. Graph represents fluorescence quantification of LAP in human macrophages (n=4; * p<0.05). (E) Ethanol-exposed human macrophages were co-treated with SIRT2 inhibitor AK-7 or DMSO ± LPS. Representative images of intracellular pHrodo bioparticles during phagocytosis and stained for LC3 to study LAP. (F) Graph represents fluorescence quantification of LAP in Ethanol-exposed human macrophages ± AK-7 ± LPS stimulation (n=4; * p<0.05).

Article Snippet: PFKP ( Cell signaling Technology, Danvers, MA, CAT# 8164S), LC3 (Cell signaling Technology, Danvers, MA, CAT# E5Q2K: western blot), LC3 (Novus Biologicals, Centennial, Colorado, CAT# NC100-2220: immunocytochemistry), SIRT2 (Cell signaling Technology, Danvers, MA, CAT# D4050), Atg4B (Cell signaling Technology, Danvers, MA, CAT# 13507S), Rubicon (Cell signaling Technology, Danvers, MA, CAT# 8465S), Beclin-1 (Cell signaling Technology, CAT# 3495S), PFKM (Abcam, Waltham, Boston, USA,CAT#ab154804), VPS34 (Abcam, Waltham, Boston, CAT# ab124905), PFKL (Santa Cruz Biotechnology, Dallas, Texas, USA, CAT# sc-393713), F4/80 (Invitrogen, Rockford, Illinois, USA, CAT# MA1-91124), Anti-rabbit Alexa Fluor 488 (Invitrogen, Rockford, Illinois, USA, CAT# A21206), Anti-rat Alexa Fluor 594 (Invitrogen, Rockford, Illinois, USA, CAT# A21209), Anti-rabbit Alexa Fluor 647 (Invitrogen, Rockford, Illinois, USA, CAT# A21245), Anti rabbit IgG control (Cell signaling Technology, Danvers, MA, CAT# 2729S), anti-mouse IgG, HRP-linked antibody (Cell signaling Technology, Danvers, MA, CAT# 7076), Anti-rabbit IgG, HRP-linked antibody (Cell signaling Technology, CAT# 7074), Acetyl lysine (Novus Biologicals, Centennial, CO, USA, CAT# NB 100-74339) pSerine (Origene, Rockville, MD, USA, CAT# AM00114PU-N), Anti- Turbo GFP (tGFP) antibody (Origene, Rockville, MD, CAT# TA150041), DDK antibody (Origene, Rockville, MD, CAT# TA50011-100), Anti-Ubiquitin antibody (EMD Millipore, Burlington, Massachusetts, USA, CAT#5-944), Biotinylated anti-SIRT2 antibody (R&D system, Minneapolis, MN, USA, CAT# BAF4358), CPA (Novus Biologicals, CAT# NBP1-30993), Beta-actin(Abcam, Waltham, Boston, CAT# ab8226), ECL (Bio-Rad, Hercules, CA, USA, CAT# 1705061).

Techniques: Expressing, Immunostaining, Fluorescence, Staining

Journal: PLoS ONE

Article Title: Sirt1 Inhibits Akt2-Mediated Porcine Adipogenesis Potentially by Direct Protein-Protein Interaction

doi: 10.1371/journal.pone.0071576

Figure Lengend Snippet: A. Sirt1 inhibits transcriptional activity of Akt2 by dose–dependent way. Please reference to Luciferase reporter assays in Materials and Methods. B. Akt2 interacts with Sirt1 in HEK293 cells. HEK293 cells were transfected with Akt2, Sirt1–Flag, or both by the calcium phosphate method. 40 h after transfection, cells were lysed, and Sirt1–Flag proteins were immunoprecipitated with agarose beads and conjugated with anti–Flag antibodies. The precipitated proteins were washed five times and analyzed by Western blotting as indicated. IP: immunoprecipitation. IB: immunoblotting. C. HEK293 cells were transfected with Akt2–Flag, Sirt2, or both by the calcium phosphate method. 40 h after transfection, cells were lysed, and Akt2–Flag proteins were immunoprecipitated with agarose beads and conjugated with anti–Flag antibodies. The precipitated proteins were washed five times and analyzed by Western blotting as indicated. D. Endogenous Akt2 in porcine adipocyte lysate was immunoprecipitated with anti–Akt2 antibody, and coprecipitation of Sirt1 was detected by Western blot assay. Endogenous Sirt1 in porcine adipocyte lysate was immunoprecipitated with anti–Sirt1 antibody, and coprecipitation of Akt2 was detected by Western blot assay. Data were represented as means ± SEM and from three independent experiments, * P <0.05, ** P <0.01.

Article Snippet: The following antibodies were used: IRS1, PI3K, phospho–PI3K (Millipore, USA), Akt1 (1∶1000), Akt2 (1∶1000), phospho–Akt2–Ser474 (1∶1000) (Bioworld, USA), Sirt1 (1∶1000), Sirt2 (1∶1000), C/EBPα (1∶1000), PPARγ (1∶500), GAPDH (1∶5000), β–actin (1∶1000) and Tubulin (1∶5000) (Santa Cruz Biotechnology, Santa Cruz, USA), FAS (1∶1000), aP2 (1∶1000), GSK3β (1∶1000), phospho-GSK3β (1∶1000) (Cell Signaling Technology, USA).

Techniques: Activity Assay, Luciferase, Transfection, Immunoprecipitation, Western Blot

Journal: Behavioural brain research

Article Title: NMDAR dependent intracellular responses associated with cocaine conditioned place preference behavior.

doi: 10.1016/j.bbr.2016.09.047

Figure Lengend Snippet: Fig. 6. NAc SIRT1 (120 kDa) (A) and SIRT2 (43 kda) (C) protein levels and correlation to CPP scores (B & D). CPu SIRT1 (120 kda) (E) and SIRT2 (43 kda) (F) protein levels and correlation

Article Snippet: Primary antibodies for pERK (9101), Research 317 (2017) 218–225 219 ERK (9102), SIRT1 (2493), SIRT2 (12672), FosB (5G4) and CREB (9197) were purchased from Cell Signaling Technologies (Beverly, MA).

Techniques:

Journal: Parkinson's Disease

Article Title: Sirtuin-2 Protects Neural Cells from Oxidative Stress and Is Elevated in Neurodegeneration

doi: 10.1155/2017/2643587

Figure Lengend Snippet: The effect of SIRT2 overexpression and inhibition was determined in toxin treated SH-SY5Y cells. SIRT2 was overexpressed in SH-SY5Y cells and control cells were transfected with empty vector following which one set of cells was treated with toxin alone and another with SIRT2 inhibitor AGK2 and toxin for 20 hours and viability measured by reduction of Alamar Blue. Toxins used: diquat (20 µ M or 10 µ M) or rotenone (20 µ M or 0.5 µ M) treated cells. Data are presented as fold- untreated (±SD) from three independent assays ( n = 3). ∗∗∗ p < 0.001 when compared to 0.2% vehicle (PBS or DMSO), one-way ANOVA (Bonferroni corrected), ### p < 0.001 and ## p < 0.01 when compared to control cells, ~~~ p < 0.001 when compared to control + AGK2 treatment, and $$ p < 0.01 and $ p < 0.05 when compared to SIRT2 cells, two-way ANOVA (Bonferroni corrected).

Article Snippet: Wild type SIRT2 (SIRT2pcDNA3.1; Plasmid number 13813) was obtained from Addgene and pcDNA 3.1 was purchased from ThermoFisher Scientific.

Techniques: Over Expression, Inhibition, Control, Transfection, Plasmid Preparation

Journal: Parkinson's Disease

Article Title: Sirtuin-2 Protects Neural Cells from Oxidative Stress and Is Elevated in Neurodegeneration

doi: 10.1155/2017/2643587

Figure Lengend Snippet: Expression of SOD2 was measured in toxin treated SH-SY5Y cells. SIRT2 was overexpressed in SH-SY5Y cells and control cells were transfected with empty vector following which one set of cells was treated with toxin alone and another with SIRT2 inhibitor AGK2 and toxin. Cells were harvested and the samples were probed for SOD2 expression. Data presented as fold-untreated (0.2% vehicle) (±SD) from three independent assays ( n = 3). ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 when compared to 0.2% vehicle, one-way ANOVA (Bonferroni corrected), ### p < 0.001 when compared to control cells, ~~~ p < 0.001 when compared to control + AGK2 treatment, and $$$ p < 0.001 and $$ p < 0.01 when compared to SIRT2, two-way ANOVA (Bonferroni corrected). M indicates molecular weight marker lane.

Article Snippet: Wild type SIRT2 (SIRT2pcDNA3.1; Plasmid number 13813) was obtained from Addgene and pcDNA 3.1 was purchased from ThermoFisher Scientific.

Techniques: Expressing, Control, Transfection, Plasmid Preparation, Molecular Weight, Marker

Journal: Parkinson's Disease

Article Title: Sirtuin-2 Protects Neural Cells from Oxidative Stress and Is Elevated in Neurodegeneration

doi: 10.1155/2017/2643587

Figure Lengend Snippet: Localisation of SIRT2 and α -synuclein in diquat treated SH-SY5Y cells. Cellular distribution of SIRT2 and phospho- α -synuclein was determined using fluorescent immunocytochemistry. Images show α -synuclein immunostaining, SIRT2 immunostaining, and all staining merged including DAPI in 20 μ M diquat treated cells. Scale bars, white scale bar = 50 μ M and red scale bar = 20 μ M; magnification: 40x. (a) represents 0.2% PBS and (b) represents 20 μ M diquat treated SH-SY5Y cells.

Article Snippet: Wild type SIRT2 (SIRT2pcDNA3.1; Plasmid number 13813) was obtained from Addgene and pcDNA 3.1 was purchased from ThermoFisher Scientific.

Techniques: Immunocytochemistry, Immunostaining, Staining

Journal: Parkinson's Disease

Article Title: Sirtuin-2 Protects Neural Cells from Oxidative Stress and Is Elevated in Neurodegeneration

doi: 10.1155/2017/2643587

Figure Lengend Snippet: Localisation of SIRT2 and α -synuclein in rotenone treated SH-SY5Y cells. Cellular distribution of SIRT2 and phospho- α -synuclein was determined using fluorescent immunocytochemistry. Images show α -synuclein immunostaining, SIRT2 immunostaining, and all staining merged including DAPI in 20 μ M rotenone treated cells. Scale bars, white scale bar = 50 μ M and red scale bar = 20 μ M; magnification: 40x. (a) represents 0.2% DMSO and (b) represents 20 μ M rotenone treated SH-SY5Y cells.

Article Snippet: Wild type SIRT2 (SIRT2pcDNA3.1; Plasmid number 13813) was obtained from Addgene and pcDNA 3.1 was purchased from ThermoFisher Scientific.

Techniques: Immunocytochemistry, Immunostaining, Staining

Journal: Parkinson's Disease

Article Title: Sirtuin-2 Protects Neural Cells from Oxidative Stress and Is Elevated in Neurodegeneration

doi: 10.1155/2017/2643587

Figure Lengend Snippet: α -Synuclein aggregate formation and quantification in toxin treated SH-SY5Y cells. SIRT2 overexpressing SH-SY5Y cells were treated with toxin (20 μ M or 10 μ M diquat or 20 μ M or 0.5 μ M rotenone) and 0.2% PBS or DMSO; cells transfected with empty vector were used as a control and another set of SIRT2 and control cells was coincubated with AGK2 and toxin. Cells were immunostained with phospho- α -synuclein. Images were captured through GFP filter under 63x magnification. The captured images represent α -synuclein staining and the bar graphs represent the aggregate quantification in diquat or rotenone treated cells. Each bar represents % α -synuclein aggregates (±SD) from three independent assays ( n = 3). ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 when compared to 0.2% vehicle, one-way ANOVA (Bonferroni corrected), ### p < 0.001, ## p < 0.01, and # p < 0.05 when compared to control cells, ~~~ p < 0.001, ~~ p < 0.01, and ~ p < 0.05 when compared to control + AGK2 treatment, and $$$ p < 0.001 and $ p < 0.05 when compared to SIRT2 cells, two-way ANOVA (Bonferroni corrected). Scale bar: 20 μ M.

Article Snippet: Wild type SIRT2 (SIRT2pcDNA3.1; Plasmid number 13813) was obtained from Addgene and pcDNA 3.1 was purchased from ThermoFisher Scientific.

Techniques: Transfection, Plasmid Preparation, Control, Staining

Journal: Parkinson's Disease

Article Title: Sirtuin-2 Protects Neural Cells from Oxidative Stress and Is Elevated in Neurodegeneration

doi: 10.1155/2017/2643587

Figure Lengend Snippet: Expression of SIRT2 in different brain regions in Parkinson's disease. The levels of SIRT2 were determined in different regions of PD patients and were compared to a control-cohort. SIRT2 band intensity was normalised with GAPDH. Data are presented as fold change (±SD) with respect to control from three independent replicates. ∗∗ p < 0.01, and ∗ p < 0.05 when compared to control; statistical analysis was done through t -test performed on GraphPad prism. Images are representative blots of SIRT2 and GAPDH. M indicates molecular weight markers lane.

Article Snippet: Wild type SIRT2 (SIRT2pcDNA3.1; Plasmid number 13813) was obtained from Addgene and pcDNA 3.1 was purchased from ThermoFisher Scientific.

Techniques: Expressing, Control, Molecular Weight

Journal: Parkinson's Disease

Article Title: Sirtuin-2 Protects Neural Cells from Oxidative Stress and Is Elevated in Neurodegeneration

doi: 10.1155/2017/2643587

Figure Lengend Snippet: Expression of SIRT2 in different regions in Parkinson's disease with Dementia. The levels of SIRT2 were determined in different regions of PDD patients and were compared to a control-cohort. SIRT2 band intensity was normalised with GAPDH. Data are presented as fold change (±SD) with respect to control from three independent replicates. ∗∗ p < 0.01 when compared to control; statistical analysis was done through t -test performed on GraphPad prism. Images are representative blots of SIRT2 and GAPDH. M indicates molecular weight marker lane.

Article Snippet: Wild type SIRT2 (SIRT2pcDNA3.1; Plasmid number 13813) was obtained from Addgene and pcDNA 3.1 was purchased from ThermoFisher Scientific.

Techniques: Expressing, Control, Molecular Weight, Marker

Journal: Parkinson's Disease

Article Title: Sirtuin-2 Protects Neural Cells from Oxidative Stress and Is Elevated in Neurodegeneration

doi: 10.1155/2017/2643587

Figure Lengend Snippet: Expression of SIRT2 in different brain regions in dementia with Lewy Bodies. The levels of SIRT2 were determined in different regions of DLB patients and were compared to a control-cohort. SIRT2 band intensity was normalised with GAPDH. Data are presented as fold change (±SD) with respect to control from three independent replicates. ∗∗ p < 0.01, and ∗ p < 0.05 when compared to control; statistical analysis was done through t -test performed on GraphPad prism. Images are representative blots of SIRT2 and GAPDH. M indicates molecular weight marker lane.

Article Snippet: Wild type SIRT2 (SIRT2pcDNA3.1; Plasmid number 13813) was obtained from Addgene and pcDNA 3.1 was purchased from ThermoFisher Scientific.

Techniques: Expressing, Control, Molecular Weight, Marker

Journal: Parkinson's Disease

Article Title: Sirtuin-2 Protects Neural Cells from Oxidative Stress and Is Elevated in Neurodegeneration

doi: 10.1155/2017/2643587

Figure Lengend Snippet: Expression of SIRT2 in different brain regions in Alzheimer's disease. The levels of SIRT2 were determined in different regions of AD patients and were compared to a control-cohort. SIRT2 band intensity was normalised with GAPDH. Data are presented as fold change (±SD) with respect to control from three independent replicates. ∗ p < 0.05 when compared to control; statistical analysis was done through t -test performed on GraphPad prism. Images are representative blots of SIRT2 and GAPDH. M indicates molecular weight marker's lane.

Article Snippet: Wild type SIRT2 (SIRT2pcDNA3.1; Plasmid number 13813) was obtained from Addgene and pcDNA 3.1 was purchased from ThermoFisher Scientific.

Techniques: Expressing, Control, Molecular Weight, Marker

Journal: Parkinson's Disease

Article Title: Sirtuin-2 Protects Neural Cells from Oxidative Stress and Is Elevated in Neurodegeneration

doi: 10.1155/2017/2643587

Figure Lengend Snippet: Total SIRT and SIRT2 activities in the frontal and temporal cortex in PD, PDD, DLB, AD, and controls. Total SIRT and SIRT2 activities were measured through a fluorometric enzymatic activity assay in the frontal and temporal cortices of PD, PDD, DLB, and AD patients and were compared to cohort-control group. ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 when compared to control, one-way ANOVA (Bonferroni corrected).

Article Snippet: Wild type SIRT2 (SIRT2pcDNA3.1; Plasmid number 13813) was obtained from Addgene and pcDNA 3.1 was purchased from ThermoFisher Scientific.

Techniques: Enzyme Activity Assay, Control

Journal: Parkinson's Disease

Article Title: Sirtuin-2 Protects Neural Cells from Oxidative Stress and Is Elevated in Neurodegeneration

doi: 10.1155/2017/2643587

Figure Lengend Snippet: Cellular distribution of SIRT2 in the temporal cortex of disease and control groups. The images show the cellular localisation of SIRT2 in grey matter of superior temporal gyrus of temporal cortex in PD, PDD, DLB, AD, and control cases. SIRT2 was localised in both in the cytoplasm and the nucleus of the neurones in the groups, being predominantly present in the cytoplasm in AD. The picture in inset is 63x oil immersion image overlaid on 10x image. Scale bars, black scale bar = 50 μ M and red scale bar = 20 μ M.

Article Snippet: Wild type SIRT2 (SIRT2pcDNA3.1; Plasmid number 13813) was obtained from Addgene and pcDNA 3.1 was purchased from ThermoFisher Scientific.

Techniques: Control