rabbit polyclonal antibody anti-sirt2 (1:1000; cat# s8447  (Millipore)

Journal: iScience

Article Title: Mitochondrial glycerol 3-phosphate dehydrogenase deficiency exacerbates lipotoxic cardiomyopathy

doi: 10.1016/j.isci.2024.109796

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-SIRT2 antibody , Proteintech , Cat# 19655-1-AP; RRID: AB_2878592.

Techniques: Virus, Recombinant, Modification, Transfection, Labeling, XF Assay, Isolation, Staining, Bicinchoninic Acid Protein Assay, Immunoprecipitation, Plasmid Preparation, Control, Software

Journal: Journal of Neuroimmune Pharmacology

Article Title: SIRT2 Inhibition Rescues Neurodegenerative Pathology but Increases Systemic Inflammation in a Transgenic Mouse Model of Alzheimer’s Disease

doi: 10.1007/s11481-023-10084-9

Figure Lengend Snippet: SIRT2 inhibition increases microglial phagocytosis of methoxy-labeled Aβ. ( a ) Experimental design for quantitative in vivo assessment of amyloid-beta phagocytic capacity and gating strategy to identify CD11b + CD45 low microglia. SSC: side scatter; FSC: forward scatter. ( b ) Quantification of Aβ phagocytosis by flow cytometry of microglia isolated from vehicle or 33i treated 8 months-old APP/PS1 mice 3 h after intraperitoneal injection of methoxy-X04 (*p < 0.05, Student’s t-test). Results are shown as mean ± SEM (n = 5–6 animals per group). ( c ) Representative FACS plots demonstrating the engulfment of Aβ by microglia isolated from APP/PS1 mice upon treatment with vehicle or 33i. Wild-type mice (WT) injected with methoxy-X04 were used to determine the methoxy-X04-threshold for non-phagocytic cells

Article Snippet: The trans-blots were blocked in TBS-Tween containing 5% powder milk for 1 h. Membranes were probed overnight at 4 °C with rabbit polyclonal antibody anti-SIRT2 (1:1000; cat# S8447, Sigma-Aldrich).

Techniques: Inhibition, Labeling, In Vivo, Flow Cytometry, Isolation, Injection

Journal: Journal of Neuroimmune Pharmacology

Article Title: SIRT2 Inhibition Rescues Neurodegenerative Pathology but Increases Systemic Inflammation in a Transgenic Mouse Model of Alzheimer’s Disease

doi: 10.1007/s11481-023-10084-9

Figure Lengend Snippet: SIRT2 inhibition induces peripheral inflammation. ( a ) Weekly body weight monitoring of WT and APP/PS1 mice during the treatment. Glucose ( b ) and Insulin ( c ) tolerance tests. 33i treatment for two months in WT and APP/PS1 mice did not have any significant effect on glucose and insulin tolerance (n = 12–14 animals per group). ( d ) Gene expression of Il-1β (F = 7.529, *p < 0.05, main effect of treatment; F = 5.532, #p < 0.05, main effect of genotype, two-way ANOVA, n = 5–6 mice per group) and ( e ) protein expression of IL-1β (F = 50.13, ***p < 0.01, main effect of treatment; F = 4.978, #p < 0.05, main effect of genotype, two-way ANOVA, n = 7–8 animals per group) in white adipose tissue of WT and APP/PS1 mice. Note that 33i treatment increased levels of this pro-inflammatory cytokine in WT and APP/PS1 animals. Peripheral gene expression of ( f ) Tnf-α (F = 5.201, *p < 0.05, main effect of treatment; F = 21.11, ###p < 0.001, main effect of genotype, two-way ANOVA) and ( g ) Tgf-β (F = 11.46, ##p < 0.01, main effect of genotype, two-way ANOVA) (n = 5–6 animals per group). 36b4 was used as an internal control. Serum levels of the cytokines ( h ) IL-6 (F = 18.76, ***p < 0.001, main effect of treatment, two-way ANOVA), ( i ) MCP-1 (F = 7.782, *p < 0.05, main effect of treatment, two-way ANOVA) and ( j ) TNF (F = 8.901, **p < 0.01, main effect of treatment, two-way ANOVA) (n = 5–8 mice per group). Results are shown as mean ± SEM

Article Snippet: The trans-blots were blocked in TBS-Tween containing 5% powder milk for 1 h. Membranes were probed overnight at 4 °C with rabbit polyclonal antibody anti-SIRT2 (1:1000; cat# S8447, Sigma-Aldrich).

Techniques: Inhibition, Expressing

Journal: Journal of Neuroimmune Pharmacology

Article Title: SIRT2 Inhibition Rescues Neurodegenerative Pathology but Increases Systemic Inflammation in a Transgenic Mouse Model of Alzheimer’s Disease

doi: 10.1007/s11481-023-10084-9

Figure Lengend Snippet: Peripheral SIRT2 inhibition impairs memory and increases systemic inflammation. ( a ) Habituation phase of the MWM. ( b ) Escape latency in the acquisition phase of the MWM and corresponding area under the curve (AUC) of the acquisition curve (F = 6.716, *p < 0.05 main effect of treatment; F = 6.580, #p < 0.05 main effect of genotype, two-way ANOVA, n = 6–8 animals per group). Note that AGK-2 treatment worsened learning capacities in both WT and APP/PS1 mice. ( c ) Representation of the percentage of time spent in the correct quadrant in the retention phase of the MWM (5 th Day: F = 4.474 *p < 0.05, main effect of treatment; 8 th Day: F = 4.854, #p < 0.05, main effect of genotype, two-way ANOVA). ( d ) Representative hippocampal sections of β-amyloid plaques stained with 6E10 antibody in brain slices (left) and amyloid burden quantification (right) in 8 months-old APP/PS1 mice treated for two months with vehicle or AGK-2 (n = 3 animals per group, 2 sections including hippocampus and frontal cortex per animal) Scale bar = 500 µm. Glucose ( e ) and Insulin ( f ) tolerance tests. No significant differences were observed between vehicle or AGK-2 treated animals (n = 5–9 mice per group). Peripheral protein expression of ( g ) IL-1β (F = 5.951, *p < 0.05, main effect of treatment, two-way ANOVA) and gene expression of ( h ) Il-1β (F = 16.33, ***p < 0.001, main effect of treatment, two-way ANOVA), ( i ) Tnf-α (F = 19.60, ***p < 0.001, main effect of treatment, two-way ANOVA) and ( j ) Tgf-β (F = 11.49, **p < 0.01, main effect of treatment, two-way ANOVA) (n = 6 animals per group). 36b4 was used as an internal control. Serum levels of the cytokines ( k ) IL-6 (F = 10.80, ***p < 0.001, main effect of treatment, two-way ANOVA), ( l ) MCP-1 and ( m ) TNF (F = 5.926, *p < 0.05, main effect of treatment, two-way ANOVA) (n = 5–8 mice per group). Results are shown as mean ± SEM

Article Snippet: The trans-blots were blocked in TBS-Tween containing 5% powder milk for 1 h. Membranes were probed overnight at 4 °C with rabbit polyclonal antibody anti-SIRT2 (1:1000; cat# S8447, Sigma-Aldrich).

Techniques: Inhibition, Staining, Expressing

Journal: Journal of Neuroimmune Pharmacology

Article Title: SIRT2 Inhibition Rescues Neurodegenerative Pathology but Increases Systemic Inflammation in a Transgenic Mouse Model of Alzheimer’s Disease

doi: 10.1007/s11481-023-10084-9

Figure Lengend Snippet: SIRT2 is increased in postmortem brain tissue from Alzheimer’s disease patients but not in serum. ( a ) Gene expression of SIRT2 in frontal cortex of postmortem control and Alzheimer’s disease (AD) human samples (*p < 0.05, Student’s t-test). β-ACTIN was used as internal control (n = 10 samples per group). ( b ) Representative western blot images (top) and SIRT2 protein levels quantification (bottom) in frontal cortex of postmortem control and AD human samples (*p < 0.05, Student’s t-test). β-ACTIN was used as internal control (n = 7 samples per group). ( c ) No significant differences between both groups were found when SIRT2 was analysed in serum samples (n = 24 samples per group)

Article Snippet: The trans-blots were blocked in TBS-Tween containing 5% powder milk for 1 h. Membranes were probed overnight at 4 °C with rabbit polyclonal antibody anti-SIRT2 (1:1000; cat# S8447, Sigma-Aldrich).

Techniques: Expressing, Western Blot

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a Western blotting was performed to examine the protein expression of SIRT2 in wild-type (WT) and SIRT2 knockout (KO) mice. b Reverse-transcription PCR (RT-PCR) was used to detect the mRNA expression of Sirt2 in WT and SIRT2 KO mice. c Immunostaining was performed to detect tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc) in MPTP-treated WT and SIRT2 KO mice (the control group was treated with normal saline (NS)). The scale bar represents 200 μm. d The number of TH-positive neurons in the SNpc was counted ( n = 3). e The open field test and f Rotarod test were used to examine MPTP-treated WT and SIRT2 KO mice ( n = 15). g RT-PCR was employed to assess the mRNA expression of α-synuclein and Sirt2 in WT mice, α-synuclein-A30P*A53T transgenic (TG) mice, and α-synuclein-A30P*A53T transgenic mice with SIRT2 knockout (TG-KO). h Immunostaining was performed to detect TH-positive neurons in the SNpc in WT, TG, and TG-KO mice. The scale bar represents 200 μm. i Numbers of TH-neurons in the SNpc were counted stereologically ( n = 3). j The open field test and k rotarod test were used to detect motor deficits in WT, TG, and TG-KO mice ( n = 8). All data are presented as the means ± SD. Statistical analyses were conducted using two-way ANOVA followed by Tukey’s post hoc test in ( d – f ). Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test in ( i – k ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: Western Blot, Expressing, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Transgenic Assay

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a The mRNA levels of SIRT2 in the SNpc were examined using RT-PCR in mice treated with normal saline (NS) or MPTP. b Quantification of SIRT2 mRNA levels shown in ( a ) ( n = 3). c The protein levels of SIRT2 in the SNpc were examined using western blotting in mice treated with NS or MPTP. d Quantification of SIRT2 protein levels shown in ( c ) ( n = 3). e – h mRNA and protein levels of SIRT2 in the SNpc assessed were assessed using RT-PCR and western blotting ( n = 3), respectively, in WT and α-synuclein-A30P*A53T transgenic (TG) mice. i – l mRNA and protein levels of SIRT2 were examined in primary culture neurons treated with 50 μM MPP + for 0, 6, 12, and 24 h using RT-PCR and western blotting ( n = 3). m – p SIRT2 expression was examined in SY5Y cells stably expressing GFP vector, GFP-α-synuclein, GFP-α-synuclein-A53T, and GFP-α-synuclein-A30P*A53T ( n = 3). All data are presented as the means ± SD. Statistical analyses were conducted using an unpaired t -test in ( b ) and ( d ). Statistical analyses were conducted using two-way ANOVA followed by Tukey’s post hoc test in ( f ) and ( h ). Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test in ( j , l , n , p ). None of the differences were significant.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transgenic Assay, Expressing, Stable Transfection, Plasmid Preparation

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a , b Subcellular localization of SIRT2 was monitored using dual immunolabeling for TH (red) and SIRT2 (green) in the SNpc in mice treated with normal saline (NS) or MPTP ( a ) as well as in WT and α-synuclein-A30P*A53T transgenic mice (TG) ( b ). c Immunofluorescence assays for SIRT2 (green) were conducted in primary culture neurons treated with 50 μM MPP + for 24 h. d The localization of SIRT2 was detected using nuclear/cytosolic immunoblotting in primary culture neurons treated with 50 μM MPP + for 0, 6, 12, and 24 h. e Quantification of SIRT2 protein levels in the cytoplasm and nucleus (separate from d ). f SH-SY5Y cells were transiently transfected with GFP vector (green) or GFP-α-synuclein-A30P*A53T (green) plasmids for 24 h and subsequently immunostained for SIRT2 (red) and observed using confocal microscopy. g Subcellular localization of SIRT2 examined using dual immunolabeling for TH (red) and SIRT2 (green) in the mouse SNpc 35 days after the striatal stereotactic injection of 2 μl (per side) normal saline (NS), or α-synuclein PFF (Abcam, ab246002, 1 μg/μl). h Localization of SIRT2 detected using nuclear/cytoplasmic immunofluorescence staining in primary culture neurons treated with 4 μg/ml PFF for 7 days. i Localization of SIRT2 detected using nuclear/cytosolic immunoblotting in primary culture neurons treated with 4 μg/ml PFF for 7 days. j Quantification of SIRT2 protein levels in the cytoplasm and nucleus (separate from [ i ]). The scale bar represents 20 μm. All data are presented as the means ± SD. Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test in ( e ). Statistical analyses were conducted using two-way ANOVA followed by Tukey’s post hoc test in ( j ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: Immunolabeling, Transgenic Assay, Immunofluorescence, Western Blot, Transfection, Plasmid Preparation, Confocal Microscopy, Injection, Staining

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a Primary culture neurons were transiently transfected with GFP vector or GFP-NLS-SIRT2 plasmids and subsequently stained using anti-SIRT2 antibodies (green), propidium iodide (PI) (red), and DAPI (blue). The scale bar represents 10 μm. b GFP-positive cells (green) that also showed positive PI staining (red) were counted as dead neurons. The percentage of PI-positive cells (red) among at least 100 GFP-positive cells was measured for each group ( n = 3). All data are presented as the means ± SD. Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test, and ** represents P < 0.01. c HEK293 cells were transfected with GFP vector and GFP-NLS-SIRT2 plasmids for 24 h and subsequently collected for RNA-seq. The differentially expressed genes between the two groups are illustrated using a heatmap ( n = 3). The colors indicate relative expression levels (red, high expression; green, low expression). d Gene Ontology (GO) terms. Frequency of sequences with assigned GO terms for the biological process category across the different samples. e KEGG pathway enrichment. Biological pathways associated with the differentially expressed genes in the different samples.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: Transfection, Plasmid Preparation, Staining, RNA Sequencing Assay, Expressing

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a Co-Immunoprecipitation (Co-IP) was performed to identify whether endogenous SIRT2 interacts with Cdk5. b A GST-pull-down assay was used to verify whether exogenous SIRT2 can bind to Cdk5. c Cultured primary neurons were pretreated with 10 μM roscovitine (ROS) for 0.5 h and subsequently treated with 50 μM MPP + for 24 h. The cytoplasmic and nuclear distribution of SIRT2 was detected using immunoblotting. d Quantification of SIRT2 protein levels in the cytoplasm and nucleus shown in c ( n = 3). e Immunofluorescence staining for SIRT2 (green) was performed to observe the distribution of SIRT2 in primary culture neurons subjected to ROS stress. The scale bar represents 10 μm. All data are presented as the means ± SD. Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test in d . * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Pull Down Assay, Cell Culture, Western Blot, Immunofluorescence, Staining

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a Liquid chromatography-mass spectrometry after a kinase assay in vitro demonstrated that the Ser331 and b Ser335 sites of SIRT2 were phosphorylated by Cdk5. c Phosphorylation levels of SIRT2 were detected in forebrain cortical tissue obtained from forebrain neuron-specific Cdk5 knockout mice and WT mice. Phosphorylation of SIRT2 was detected in primary culture neurons using immunoprecipation and western blotting with anti-phosS/TP and anti-SIRT2 antibodies. d SDS-PAGE used for the Cdk5/p35 kinase assay in vitro. Purified GST-SIRT2 WT, S331A, S335A, and S331AS335A (AA) fusion proteins were mixed with active Cdk5/p35 and ATP, and the reaction mixture was analyzed using western blotting with anti-phosS/TP and anti-GST antibodies. e HEK293 cells were transiently transfected with Flag-SIRT2 WT or Flag-SIRT2 mutation DD (double mutations at S331D and S335D) plasmids for 24 h, and subsequently, the cytoplasmic and nuclear distribution of SIRT2 (green) was detected using immunofluorescence staining. The scale bar represents 10 μm. f Immunoblotting assays for SIRT2 was performed using an anti-Flag antibody in HEK293 cells transfected with Flag-SIRT2 WT or Flag-SIRT2-DD. g Quantification of SIRT2 protein levels shown in f ( n = 3). All data are presented as the means ± SD. Statistical analyses were conducted using two-way ANOVA followed by Tukey’s post hoc test in g . * P < 0.05, *** P < 0.001.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: Liquid Chromatography, Mass Spectrometry, Kinase Assay, In Vitro, Knock-Out, Western Blot, SDS Page, Purification, Transfection, Mutagenesis, Immunofluorescence, Staining

Journal: NPJ Parkinson's Disease

Article Title: Cdk5 phosphorylation-induced SIRT2 nuclear translocation promotes the death of dopaminergic neurons in Parkinson’s disease

doi: 10.1038/s41531-022-00311-0

Figure Lengend Snippet: a Primary culture neurons were pretreated with the SIRT2 328–339 or Scramble peptide conjugated with Myristic acid (Myr) and subsequently treated with 100 μM MPP + for 48 h. The survival percentage of neurons was detected using an MTT assay. b , c Immunostaining for TH in the SNpc, d the open field test, and e the rotarod test were performed in MPTP-treated mice who received Myr-SIRT2 328–339 at a dose of 2 mg/kg (i.p.) once per day. The scramble peptide was used as the control. The number of TH-positive neurons in the SNpc was counted (Myr-SIRT2 328–339 , n = 3; Scramble, n = 4). The behavioral tests were conducted in 10 (Myr-SIRT2 328–339 ) and 12 mice (Scramble) each. All data are presented as the means ± SD. Statistical analyses were conducted using one-way ANOVA followed by Tukey’s post hoc test in ( a , c – e ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The rabbit polyclonal anti-Sirt2 antibody (s8447, WB-1:4000, IF-1:200) was purchased from Sigma-Aldrich.

Techniques: MTT Assay, Immunostaining

Journal: Scientific Reports

Article Title: SIRT2 promotes murine melanoma progression through natural killer cell inhibition

doi: 10.1038/s41598-021-92445-z

Figure Lengend Snippet: Systemic SIRT2 overexpression increases growth of melanoma tumors in mice. ( a ) Gross specimens of subcutaneous melanoma tumors harvested from Sirt2 -KI and WT mice inoculated with 1 × 10 5 B16-F10 melanoma cells. ( b ) Tumors in WT (n = 14) and Sirt2- KI (n = 16) mice were harvested on Day 20 and weighed. Student’s t test. ( c ) Tumor progression and size were monitored 20 days post injection in WT and Sirt2 -KI mice (n = 3). Two-way ANOVA with Bonferroni correction. ( d ) Western blot showing decreased SIRT2 expression in Sirt2 -KO B16-F10 cells following Sirt2 knockout via CRISPR gene editing. Sirt2 -OE B16-F10 cells overexpress SIRT2 as demonstrated by western blot following transfection with Flag-WT- Sirt2. ( e ) C57BL/6 WT mice were injected with WT, Sirt2- OE, and Sirt2- KO B16-F10 melanoma cells and tumor development and growth were monitored for 14 days. No significant difference in tumor progression was observed between the cell groups. n = 10. Two-way ANOVA with post-hoc Bonferroni correction. Data points are mean values ± SEM.* P < 0.05 and **** P < 0.0001.

Article Snippet: The antibodies used and their dilutions were: Anti-SIRT2 rabbit polyclonal antibody (Proteintech Group, Inc, Rosemont, IL, USA), 1:500; anti-Flag mouse monoclonal antibody (Sigma, St. Louis, MO), 1:1000; anti-acetyl-α-tubulin Lys40 mouse monoclonal antibody and anti-vinculin rabbit monoclonal antibody (Cell Signaling Technology, Inc), 1:1000.

Techniques: Over Expression, Injection, Western Blot, Expressing, Knock-Out, CRISPR, Transfection

Journal: Scientific Reports

Article Title: SIRT2 promotes murine melanoma progression through natural killer cell inhibition

doi: 10.1038/s41598-021-92445-z

Figure Lengend Snippet: Natural killer cell distribution is altered by SIRT2 expression. ( a ) Subcutaneous melanoma tumors from WT and Sirt2 -KI mice were harvested and analyzed by flow cytometry for the prevalence of NK cells among leukocytes. The gating strategy was designed to isolate NK cells by selecting for cells that are NKp46 positive and CD3E negative. ( b ) The percentage of tumor-infiltrating leukocytes that expressed the NK cell signature were quantified in WT (n = 22) and Sirt2 -KI mice (n = 11). ( c ) Absolute quantities of NK cells are illustrated in Sirt2 -KI and WT mice (n = 6). Data points represent mean values with error bars representing the SEM. Student’s t test, *P < 0.05 and *** P < 0.001.

Article Snippet: The antibodies used and their dilutions were: Anti-SIRT2 rabbit polyclonal antibody (Proteintech Group, Inc, Rosemont, IL, USA), 1:500; anti-Flag mouse monoclonal antibody (Sigma, St. Louis, MO), 1:1000; anti-acetyl-α-tubulin Lys40 mouse monoclonal antibody and anti-vinculin rabbit monoclonal antibody (Cell Signaling Technology, Inc), 1:1000.

Techniques: Expressing, Flow Cytometry

Journal: Scientific Reports

Article Title: SIRT2 promotes murine melanoma progression through natural killer cell inhibition

doi: 10.1038/s41598-021-92445-z

Figure Lengend Snippet: SIRT2 overexpression suppresses NK cell function ex vivo. NK cells were harvested from the spleens of Sirt2- KI (n = 14) and WT (n = 15) mice not bearing tumors. ( a ) NK cells were isolated and quantified by selecting for cells that are NKp46 positive and CD3E negative. Student’s t test. ( b ) Splenic NK cells were co-incubated with B16-F10 melanoma cells at E:T ratios ranging from 6.25:1 to 50:1. The cytotoxic activity of splenic NK cells is represented by % specific lysis. n = 10 and 9 for NK cells from WT and Sirt2 -KI mice, respectively. Two-way ANOVA with Bonferroni correction. ( c ) RT-qPCR was utilized to determine the mRNA expression of cytokines related to NK cell activity. Multiple Student’s t test. n = 3 and 4. Data points are mean values ± SEM. *P < 0.05 and **P < 0.01.

Article Snippet: The antibodies used and their dilutions were: Anti-SIRT2 rabbit polyclonal antibody (Proteintech Group, Inc, Rosemont, IL, USA), 1:500; anti-Flag mouse monoclonal antibody (Sigma, St. Louis, MO), 1:1000; anti-acetyl-α-tubulin Lys40 mouse monoclonal antibody and anti-vinculin rabbit monoclonal antibody (Cell Signaling Technology, Inc), 1:1000.

Techniques: Over Expression, Cell Function Assay, Ex Vivo, Isolation, Incubation, Activity Assay, Lysis, Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: SIRT2 promotes murine melanoma progression through natural killer cell inhibition

doi: 10.1038/s41598-021-92445-z

Figure Lengend Snippet: SIRT2 expression regulates human NK cell proliferation and function in vitro. ( a ) SIRT2 expression was modified through transfection and expression of exogenous Sirt2 in human NK-92MI cells as demonstrated in the western blot. ( b ) Cell proliferation assay of NK-92MI cells with ( Sirt2 -OE (n = 4)) and without (control (n = 5)) exogenous Sirt2 . Cell counts were performed every 24 h for a total of 96 h. Two-way ANOVA with Bonferroni correction. ( c ) Chemotaxis was evaluated using a transwell migration assay. The migration index was determined by the number of transmigrated cells after 4 h. (n = 3). ( d ) NK-92MI cells were co-incubated with K562 leukemia cells at E:T ratios ranging from 3.125:1 to 100:1. The cytotoxic activity of NK-92MI cells is represented by % specific lysis. n = 4. Two-way ANOVA with Bonferroni correction. ( e ) RT-qPCR was utilized to determine the expression of multiple NK cell cytokines following induction by IL-12. Multiple Student’s t test. n = 3. Data points are mean values ± SEM. *** P < 0.001 and **** P < 0.0001 .

Article Snippet: The antibodies used and their dilutions were: Anti-SIRT2 rabbit polyclonal antibody (Proteintech Group, Inc, Rosemont, IL, USA), 1:500; anti-Flag mouse monoclonal antibody (Sigma, St. Louis, MO), 1:1000; anti-acetyl-α-tubulin Lys40 mouse monoclonal antibody and anti-vinculin rabbit monoclonal antibody (Cell Signaling Technology, Inc), 1:1000.

Techniques: Expressing, In Vitro, Modification, Transfection, Western Blot, Proliferation Assay, Control, Chemotaxis Assay, Transwell Migration Assay, Migration, Incubation, Activity Assay, Lysis, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: SIRT2 promotes murine melanoma progression through natural killer cell inhibition

doi: 10.1038/s41598-021-92445-z

Figure Lengend Snippet: The effect of SIRT2 on tumor progression is mediated through NK cells. WT (n = 6 and 5) and Sirt2 -KI (n = 7 and 5) mice were divided into two groups with one receiving anti-AsGM1 to deplete NK cell populations, and the other was treated with saline. Following inoculation with B16-F10 melanoma cells, tumors were monitored for growth and harvested after 21 days. Data points represent mean values ± SEM. One-way ANOVA with post-hoc Tukey HSD test, ***P < 0.001.

Article Snippet: The antibodies used and their dilutions were: Anti-SIRT2 rabbit polyclonal antibody (Proteintech Group, Inc, Rosemont, IL, USA), 1:500; anti-Flag mouse monoclonal antibody (Sigma, St. Louis, MO), 1:1000; anti-acetyl-α-tubulin Lys40 mouse monoclonal antibody and anti-vinculin rabbit monoclonal antibody (Cell Signaling Technology, Inc), 1:1000.

Techniques: Saline

Journal: Scientific Reports

Article Title: SIRT2 promotes murine melanoma progression through natural killer cell inhibition

doi: 10.1038/s41598-021-92445-z

Figure Lengend Snippet: Pharmacological inhibition of SIRT2 enhances NK cell function and suppresses tumor progression. ( a ) Western blot showing decreased SIRT2 expression and deacetylase activity, as indicated by increased levels of α-tubulin K40 acetylation, in C57BL/6 mice treated with SirReal2, a small molecule SIRT2 inhibitor (n = 3). ( b ) Tumor progression and size were monitored up to 19 days following injection of B16-F10 cells into WT and Sirt2- KO mice treated with SirReal2 or vehicle (n = 12 and 18 for WT and Sirt2 -KO). Two-way ANOVA with Bonferroni correction. ( c ) Tumors were harvested on Day 19 and weighed. ( d ) Subcutaneous melanoma tumors from WT mice were harvested and analyzed by flow cytometry for the prevalence of NK cells among leukocytes. The gating strategy was designed to identify NK cells as cells that are NKp46 positive and CD3E negative. ( e ) Abundance of NK cells as a percent of total leukocytes infiltrating melanoma tumors in WT (n = 10 and 8) and Sirt2 -KO (n = 5) mice treated with vehicle or SirReal2 was quantified. ( f ) Absolute number of tumor-infiltrating NK cells in Sirt2 -KI and WT mice. ( c , e , f ) One-way ANOVA with post-hoc Tukey HSD. Data points are mean values ± SEM. *** P < 0.001 and **** P < 0.0001.

Article Snippet: The antibodies used and their dilutions were: Anti-SIRT2 rabbit polyclonal antibody (Proteintech Group, Inc, Rosemont, IL, USA), 1:500; anti-Flag mouse monoclonal antibody (Sigma, St. Louis, MO), 1:1000; anti-acetyl-α-tubulin Lys40 mouse monoclonal antibody and anti-vinculin rabbit monoclonal antibody (Cell Signaling Technology, Inc), 1:1000.

Techniques: Inhibition, Cell Function Assay, Western Blot, Expressing, Histone Deacetylase Assay, Activity Assay, Injection, Flow Cytometry

Journal: Scientific Reports

Article Title: SIRT2 promotes murine melanoma progression through natural killer cell inhibition

doi: 10.1038/s41598-021-92445-z

Figure Lengend Snippet: The dual role of SIRT2 in cancer initiation and progression. An illustration of SIRT2’s potential roles in cancer initiation and progression are shown with treatment strategies targeting SIRT2 proposed.

Article Snippet: The antibodies used and their dilutions were: Anti-SIRT2 rabbit polyclonal antibody (Proteintech Group, Inc, Rosemont, IL, USA), 1:500; anti-Flag mouse monoclonal antibody (Sigma, St. Louis, MO), 1:1000; anti-acetyl-α-tubulin Lys40 mouse monoclonal antibody and anti-vinculin rabbit monoclonal antibody (Cell Signaling Technology, Inc), 1:1000.

Techniques: