primary antibody against sirt6  (Cell Signaling Technology Inc)

Journal: Nature Communications

Article Title: Sirt6 deficiency in mast cells promotes adipose fibroinflammation in obesity through galectin-3 signaling

doi: 10.1038/s41467-025-66040-z

Figure Lengend Snippet: a Representative immunofluorescence staining of mast cells in omental adipose tissue from individuals with a BMI < 25 (lean), 25 ≤ BMI < 30 (overweight), and BMI ≥ 30 (obesity). Both total and degranulated mast cells were counted ( n = 13 for lean, n = 16 for overweight, n = 25 for obese). b Adipose-derived mast cells (FcεRI + CD117 + ) from the SVF were isolated from human omental adipose tissues, and Sirt6 protein levels were compared among individuals with a BMI < 25, 25 ≤ BMI < 30, and BMI ≥ 30 ( n = 25 for lean, n = 14 for overweight, n = 6 for obese). c A scatter plot was created to compare mast cell Sirt6 expression with body mass index (BMI, n = 45), plasma leptin ( n = 44), and plasma adiponectin ( n = 44) levels. d , e C57BL/6 mice were fed either a normal chow diet (NCD) or a high-fat diet (HFD) for 3 or 16 weeks. Epididymal adipose tissues were then harvested for western blot analysis of Sirt6 in mast cells ( d ) or for histological analysis with toluidine blue staining to identify mast cells ( e ). Both total and degranulated mast cells were counted ( n = 14 for NCD, n = 15 for HFD). Values are presented as mea n ± SD. One-way ANOVA followed by Sidak’s multiple comparisons analysis (a, b) and unpaired two-tailed t test between two groups ( e ) were conducted for statistical analyses. Pearson correlation coefficients were calculated between continuous variables in two-sided distributions ( c ). Source data are provided as a Source Data file.

Article Snippet: Chromatin was immunoprecipitated overnight at 4 °C with antibodies against Sirt6 (#12486, 1:250 dilution), NF-κB (#8242, 1:250 dilution), nonspecific IgG (#2729, 1:250 dilution, all from Cell Signaling Technology), and Ac-H3K9 (#H9286, 1:250 dilution, Sigma-Aldrich). qPCR was performed on ChIP DNA to assess the occupancy of Sirt6 or Ac-H3K9 at the Lgals3 and Lgals9 promoters.

Techniques: Immunofluorescence, Staining, Derivative Assay, Isolation, Expressing, Clinical Proteomics, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Sirt6 deficiency in mast cells promotes adipose fibroinflammation in obesity through galectin-3 signaling

doi: 10.1038/s41467-025-66040-z

Figure Lengend Snippet: a Cma1-Cre (Cre), WT ( Sirt6 fl/fl , fl/fl) and KO ( Sirt6 -/- , -/-) mice were fed a HFD for 16 weeks from the age of 6 weeks and metabolically assessed. b , c Body weight changes (b, n = 8 per group) and food intake (c, n = 8 per group). d , e Glucose concentrations during an intraperitoneal glucose tolerance ( d , n = 8 per group) and an insulin tolerance test ( e , n = 8 per group) were measured, and areas under the curve were compared. f Fat and liver tissue weights at the end of study ( n = 8 per group). g Adipose tissue sections were H&E stained, and adipocyte size in H&E sections was quantified ( n = 52 for Cre, n = 55 for WT, n = 55 for KO). Values are presented as mean ± SD. Values on the line in panels ( b , d , e ) represent statistical comparisons with the Sirt6 fl/fl group. Two-way ANOVA followed by Bonferroni’s post hoc analysis ( b – e ) and one-way ANOVA followed by Sidak’s multiple comparisons analysis was conducted for statistical analyses ( f , g ). Source data are provided as a Source Data file. Panel ( a ) was created in BioRender. H, J. (2025) https://BioRender.com/lh1p3yb .

Article Snippet: Chromatin was immunoprecipitated overnight at 4 °C with antibodies against Sirt6 (#12486, 1:250 dilution), NF-κB (#8242, 1:250 dilution), nonspecific IgG (#2729, 1:250 dilution, all from Cell Signaling Technology), and Ac-H3K9 (#H9286, 1:250 dilution, Sigma-Aldrich). qPCR was performed on ChIP DNA to assess the occupancy of Sirt6 or Ac-H3K9 at the Lgals3 and Lgals9 promoters.

Techniques: Metabolic Labelling, Staining

Journal: Nature Communications

Article Title: Sirt6 deficiency in mast cells promotes adipose fibroinflammation in obesity through galectin-3 signaling

doi: 10.1038/s41467-025-66040-z

Figure Lengend Snippet: a . Schematic illustration of the transfer of WT or Sirt6-deficient adipose-derived mast cells (ADMCs, 2 ×10 6 cells) into mast cell-deficient Kit W-sh/W-sh mice. b - e . Body weights changes over time (b, n = 7 per group), wet weight of epididymal (EAT), inguinal (IAT), brown adipose tissue (BAT), and liver (c, n = 7 per group), glucose concentrations during an intraperitoneal glucose tolerance (d, n = 7 per group) and an insulin tolerance test (e, n = 7 per group) were measured. Areas under the curve during glucose and insulin tolerance tests were compared. f . EAT sections were stained with H&E or immunostained with antibodies against tryptase. Adipocyte size in H&E-stained sections ( n = 37 per group) and total and degranulated mast cell numbers in tryptase immunostained sections ( n = 4 per group) were determined. Values are presented as mean ± SD. Red and blue values on the line in panels b, d, and e indicate statistical comparisons with the Sirt6 fl/fl mast cell group and PBS group, respectively. One-way ANOVA followed by Sidak’s multiple comparisons analysis (c, f) and two-way ANOVA followed by Bonferroni’s post hoc analysis (b, d, e) were conducted for statistical analyses Source data are provided as a Source Data file. Panel (a) was created in BioRender. H, J. (2025) https://BioRender.com/49artbl .

Article Snippet: Chromatin was immunoprecipitated overnight at 4 °C with antibodies against Sirt6 (#12486, 1:250 dilution), NF-κB (#8242, 1:250 dilution), nonspecific IgG (#2729, 1:250 dilution, all from Cell Signaling Technology), and Ac-H3K9 (#H9286, 1:250 dilution, Sigma-Aldrich). qPCR was performed on ChIP DNA to assess the occupancy of Sirt6 or Ac-H3K9 at the Lgals3 and Lgals9 promoters.

Techniques: Derivative Assay, Staining

Journal: Nature Communications

Article Title: Sirt6 deficiency in mast cells promotes adipose fibroinflammation in obesity through galectin-3 signaling

doi: 10.1038/s41467-025-66040-z

Figure Lengend Snippet: a , b mRNA levels (a, n = 6 per group) in EAT tissues and protein levels (b, n = 6 per group) in plasma for galectin-1, galectin-3 and galectin-9 in WT and KO mice. c Heatmap displaying the ligand-receptor signaling network of the galectin pathway between immune cell types from WT and KO mice. Each cell in the heatmap represents the predicted communication probability from a sender to a receiver cell type. The bar plot above the heatmap quantifies the total outgoing signaling strength of each cell type, while the bar plot to the right reflects the total incoming signaling strength. TCP, total communication probability. d Bone marrow-derived mast cells (BMMCs) were transfected with control siRNA (siCtrl) or Sirt6 siRNA (siSirt6). Successful Sirt6 silencing was confirmed by qPCR and western blotting ( n = 6 per group). e – i Lgals3 and Lgals9 mRNA levels (e, n = 6 per group), RELA (p65 subunit of NF-κB) binding to the promoters of Lgals3 and Lgals9 ( f , n = 5 per group), Sirt6 binding to the promoters of Lgals3 and Lgasl9 ( g , n = 5 per group), enrichment of Ac-H3K9 on the Lgals3 and Lgals9 promoters ( h , n = 4 per group), and acetylation of H3K9 ( i , n = 3 per group) were compared between control and Sirt6 silenced BMMCs. j , k . Plasma levels of galectin-3 and galectin-9 in human subjects were measured by ELISA ( n = 24 for lean, n = 14 for overweight, n = 6 for obese). A scatter plot was created to examine the correlation between mast cell Sirt6 protein levels and plasma galectin-3 and galectin-9 levels. Values are presented as mean ± SD. Unpaired two-tailed t test between the two groups ( a , b , d , e , i ), one-way ANOVA followed by Sidak’s multiple comparisons analysis ( j , k ) and two-way ANOVA followed by Bonferroni’s post hoc analysis were conducted for statistical analyses ( f – h ). Pearson correlation coefficients were calculated between continuous variables in two-sided distributions ( j , k). Source data are provided as a Source Data file.

Article Snippet: Chromatin was immunoprecipitated overnight at 4 °C with antibodies against Sirt6 (#12486, 1:250 dilution), NF-κB (#8242, 1:250 dilution), nonspecific IgG (#2729, 1:250 dilution, all from Cell Signaling Technology), and Ac-H3K9 (#H9286, 1:250 dilution, Sigma-Aldrich). qPCR was performed on ChIP DNA to assess the occupancy of Sirt6 or Ac-H3K9 at the Lgals3 and Lgals9 promoters.

Techniques: Clinical Proteomics, Derivative Assay, Transfection, Control, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Sirt6 deficiency in mast cells promotes adipose fibroinflammation in obesity through galectin-3 signaling

doi: 10.1038/s41467-025-66040-z

Figure Lengend Snippet: a Schematic illustration of the adoptive transfer of WT or Sirt6-deficient adipose-derived mast cells (ADMCs), infected with lentivirus carrying shRNA targeting either control shRNA (Lv-eGFP-shCtrl, shCtrl) or galectin-3 (Lv-eGFP-shLgals3, shLgals3), into Kit W-sh/W-sh mice. b – d Body weights during 16 weeks of HFD ( b , n = 6 per group), glucose concentrations during an intraperitoneal glucose tolerance ( c , n = 6 per group) and an insulin tolerance test ( d , n = 6 per group). Areas under the curve during glucose and insulin tolerance tests were compared. Red and pink values on the line in panels ( b , c , and d ) indicate statistical comparisons with the Sirt6 fl/fl MC+shCtrl and Sirt6 -/- MC+shCtrl group, respectively. e EAT sections were stained with H&E or Sirius Red, or immunostained with anti-F4/80 antibody. Representative images and quantification results of adipocyte size in H&E-stained sections ( n = 37 per group) and macrophage numbers in F4/80-immunostained sections ( n = 7 per group) are shown. f Hydroxyproline content in EAT of mice as a measure of fibrosis ( f , n = 6 per group). g EAT sections were immunostained for tryptase, and total and degranulated mast cell counts ( n = 4 per group) were determined ( g ). Values are presented as mean ± SD. Two-way ANOVA followed by Bonferroni’s post hoc analysis ( b – d ) and one-way ANOVA followed by Sidak’s multiple comparisons analysis ( e – g ) were conducted for statistical analyses. Source data are provided as a Source Data file. Panel ( a ) was created in BioRender. H, J. (2025) https://BioRender.com/swlgc9x .

Article Snippet: Chromatin was immunoprecipitated overnight at 4 °C with antibodies against Sirt6 (#12486, 1:250 dilution), NF-κB (#8242, 1:250 dilution), nonspecific IgG (#2729, 1:250 dilution, all from Cell Signaling Technology), and Ac-H3K9 (#H9286, 1:250 dilution, Sigma-Aldrich). qPCR was performed on ChIP DNA to assess the occupancy of Sirt6 or Ac-H3K9 at the Lgals3 and Lgals9 promoters.

Techniques: Adoptive Transfer Assay, Derivative Assay, Infection, shRNA, Control, Staining

Journal: Cell Death & Disease

Article Title: Impaired SUMOylation of FoxA1 promotes nonalcoholic fatty liver disease through down-regulation of Sirt6

doi: 10.1038/s41419-024-07054-1

Figure Lengend Snippet: A RT-qPCR analysis of Sirt6, Acox1, Lpl, Pparα, Cpt1a, and Cyp4a14 levels in the liver tissues of mice. B The protein levels of Sirt6, FoxO1, Ac-FoxO1, Pparα, Acox1, Lpl, Cpt1a, and Cyp4a14 in livers were determined by Western blotting. C Primary hepatocytes were isolated from FoxA1-LKO mice and infected with Ad-LacZ or Ad-FoxA1 before treatment with palmitic acid (PAL) (0.4 mM). Lipid droplet formation was evaluated by BODIPY staining (scale bar = 100 µm). D , E RT-qPCR and Western blotting analyzed Sirt6, FoxO1, Ac-FoxO1, Pparα, Acox1, Lpl, Cpt1a and Cyp4a14 expression in the primary hepatocytes. F , G Primary hepatocytes were isolated from FoxA1 flox/flox mice and infected with Ad-shLacZ or Ad-shFoxA1, followed by treatment with PAL. Sirt6, FoxO1, Ac-FoxO1, Pparα, Acox1, Lpl, Cpt1a, and Cyp4a14 expression levels were measured by RT-qPCR and Western blotting. H , I Primary hepatocytes from FoxA1 flox/flox mice were infected with Ad-FoxA1, Ad-shSirt6, or a combination of them, and then treated with PAL. Sirt6, FoxO1, Ac-FoxO1, Pparα, Acox1, Lpl, Cpt1a, and Cyp4a14 expression levels were detected by RT-qPCR and Western blotting. Data was repeated at least 3 times. * p < 0.05, ** p < 0.01, and *** p < 0.001. For A-G, Student’s t test was performed. For H-I, one-way ANOVA followed by Tukey’s multiple comparison test was performed.

Article Snippet: The blots were blocked by skim milk and incubated using the primary antibodies against Sirt6 (A18468, 1:500, ABclonal), Cpt1a (ab234111, 1:1000, Abcam), Acox1 (ab184032, 1:1000, Abcam), Lpl (ab91606, 1:1000, Abcam), Pparα (bs-3614R, 1:500, Bioss), Cyp4a14 (ab3573, 1:1000, Abcam), FoxA1 (A15278, 1:500, ABclonal), Sae1 (ab185949, 1:1000, Abcam), Sae2 (ab185955, 1:1000, Abcam), Ubc9 (ab33044, 1:1000, Abcam), Pias1/2 (ab77231, 1:1000, Abcam), Pias3 (ab105178, 1:1000, Abcam), Pias4 (ab137500, 1:1000, Abcam), FoxO1 (ab70382, 1:2000, Abcam), Ac-FoxO1 (PA5-104560, 1:1000, Thermo Fisher), Ac-K (ab190479, 1:1000, Abcam), Cpt2 (ab181114, 1:1000, Abcam), β-actin (bs-0061R, 1:5000, Bioss) at 4 °C overnight.

Techniques: Quantitative RT-PCR, Western Blot, Isolation, Infection, Staining, Expressing, Comparison

Journal: Cell Death & Disease

Article Title: Impaired SUMOylation of FoxA1 promotes nonalcoholic fatty liver disease through down-regulation of Sirt6

doi: 10.1038/s41419-024-07054-1

Figure Lengend Snippet: Primary hepatocytes extracted from FoxA1-LKO mice were transduced with Ad-LacZ or Ad-Sirt6, followed by PAL treatment. A Primary hepatocytes were received BODIPY staining to observe lipid droplets (scale bar = 100 µm). After feeding with HFD for 2 weeks, Ad-LacZ were injected into FoxA1 flox/flox mice, and Ad-LacZ or Ad-Sirt6 were injected into FoxA1-LKO mice. 14 days after injection, the liver tissues were collected. B , C Sirt6, FoxO1, Ac-FoxO1, Pparα, Acox1 and Lpl levels were detected by RT-qPCR and Western blotting, respectively. D , E Liver cholesterol and triglycerides levels were determined. F Liver steatosis was evaluated by HE staining (scale bar = 50 µm). G Lipid accumulation in livers was analyzed by Oil-Red O staining (scale bar = 50 µm). N = 6, * p < 0.05, ** p < 0.01, and *** p < 0.001. One-way ANOVA followed by Tukey’s multiple comparison test was performed.

Article Snippet: The blots were blocked by skim milk and incubated using the primary antibodies against Sirt6 (A18468, 1:500, ABclonal), Cpt1a (ab234111, 1:1000, Abcam), Acox1 (ab184032, 1:1000, Abcam), Lpl (ab91606, 1:1000, Abcam), Pparα (bs-3614R, 1:500, Bioss), Cyp4a14 (ab3573, 1:1000, Abcam), FoxA1 (A15278, 1:500, ABclonal), Sae1 (ab185949, 1:1000, Abcam), Sae2 (ab185955, 1:1000, Abcam), Ubc9 (ab33044, 1:1000, Abcam), Pias1/2 (ab77231, 1:1000, Abcam), Pias3 (ab105178, 1:1000, Abcam), Pias4 (ab137500, 1:1000, Abcam), FoxO1 (ab70382, 1:2000, Abcam), Ac-FoxO1 (PA5-104560, 1:1000, Thermo Fisher), Ac-K (ab190479, 1:1000, Abcam), Cpt2 (ab181114, 1:1000, Abcam), β-actin (bs-0061R, 1:5000, Bioss) at 4 °C overnight.

Techniques: Transduction, Staining, Injection, Quantitative RT-PCR, Western Blot, Comparison

Journal: Cell Death & Disease

Article Title: Impaired SUMOylation of FoxA1 promotes nonalcoholic fatty liver disease through down-regulation of Sirt6

doi: 10.1038/s41419-024-07054-1

Figure Lengend Snippet: The primary hepatocytes were treated with 0.4 mM palmitic acid (PAL) for 48 h. A The sites of SUMOylated FoxA1 was predicted by sumoplot. B Western blotting analysis of Sae1, Sae2, Ubc9, Pias1/2, Pias3, Pias4 protein levels in primary hepatocytes. C Ni 2+ -NTA pull-down assay was performed to validate the interaction between FoxA1 and Sumo1, Sumo2, or Sumo3 in HEK293T cells. D Ni 2+ -NTA pull-down assay was performed to validate the interaction between FoxA1 and Sumo2 in AML-12 cells. E The direct binding of Sumo2 to FoxA1 protein in primary hepatocytes was evaluated by Co-IP. Mice were subjected to regular chow or HFD feeding for 10 weeks. F FoxA1 SUMOylation level in livers was detected by Co-IP assay. Lipid droplet formation in primary hepatocytes was observed by Oil red O staining ( G ) and BODIPY staining ( H ) (scale bar = 100 µm). I Western blotting analysis of protein levels of Sirt6 and Pparα in primary hepatocytes. Data was repeated at least 3 times. ** p < 0.01. Student’s t test was performed.

Article Snippet: The blots were blocked by skim milk and incubated using the primary antibodies against Sirt6 (A18468, 1:500, ABclonal), Cpt1a (ab234111, 1:1000, Abcam), Acox1 (ab184032, 1:1000, Abcam), Lpl (ab91606, 1:1000, Abcam), Pparα (bs-3614R, 1:500, Bioss), Cyp4a14 (ab3573, 1:1000, Abcam), FoxA1 (A15278, 1:500, ABclonal), Sae1 (ab185949, 1:1000, Abcam), Sae2 (ab185955, 1:1000, Abcam), Ubc9 (ab33044, 1:1000, Abcam), Pias1/2 (ab77231, 1:1000, Abcam), Pias3 (ab105178, 1:1000, Abcam), Pias4 (ab137500, 1:1000, Abcam), FoxO1 (ab70382, 1:2000, Abcam), Ac-FoxO1 (PA5-104560, 1:1000, Thermo Fisher), Ac-K (ab190479, 1:1000, Abcam), Cpt2 (ab181114, 1:1000, Abcam), β-actin (bs-0061R, 1:5000, Bioss) at 4 °C overnight.

Techniques: Western Blot, Pull Down Assay, Binding Assay, Co-Immunoprecipitation Assay, Staining

Journal: Cell Death & Disease

Article Title: Impaired SUMOylation of FoxA1 promotes nonalcoholic fatty liver disease through down-regulation of Sirt6

doi: 10.1038/s41419-024-07054-1

Figure Lengend Snippet: Primary hepatocytes and AML-12 cells were infected with Ad-FoxA1, and then stimulated with 0.4 mM PAL for 48 h. A FoxA1 and Sirt6 protein levels were measured by Western blotting. B JASPAR database predicted the binding sites of FoxA1 to Sirt6 promoter. C ChIP assay validated the direct interaction between FoxA1 and Sirt6 promoter. D The transcription activity of Sirt6 was detected by dual-luciferase reporter assay. PAL-treated primary hepatocytes were further administrated with SUMOylation inhibitor (GA, 10 nM) or activator (N106, 10 μM). E FoxA1 SUMOylation was assessed by the SUMOylation Assay Ultra Kit. F Ni-NTA pull-down assay was performed to determine the interaction between FoxA1 and Sumo2. G Western blotting analysis of Sirt6, Pparα, and Cpt2 protein levels and RT-qPCR analysis of Pparα, Cpt2 and Cpt1a in primary hepatocytes. H Lipid droplet formation was evaluated by BODIPY staining (scale bar = 100 µm). Data was repeated at least 3 times. * p < 0.05, ** p < 0.01, and *** p < 0.001. For ( C ), Student’s t test was performed. For ( A , D , E , G , H ), one-way ANOVA followed by Tukey’s multiple comparison test was performed.

Article Snippet: The blots were blocked by skim milk and incubated using the primary antibodies against Sirt6 (A18468, 1:500, ABclonal), Cpt1a (ab234111, 1:1000, Abcam), Acox1 (ab184032, 1:1000, Abcam), Lpl (ab91606, 1:1000, Abcam), Pparα (bs-3614R, 1:500, Bioss), Cyp4a14 (ab3573, 1:1000, Abcam), FoxA1 (A15278, 1:500, ABclonal), Sae1 (ab185949, 1:1000, Abcam), Sae2 (ab185955, 1:1000, Abcam), Ubc9 (ab33044, 1:1000, Abcam), Pias1/2 (ab77231, 1:1000, Abcam), Pias3 (ab105178, 1:1000, Abcam), Pias4 (ab137500, 1:1000, Abcam), FoxO1 (ab70382, 1:2000, Abcam), Ac-FoxO1 (PA5-104560, 1:1000, Thermo Fisher), Ac-K (ab190479, 1:1000, Abcam), Cpt2 (ab181114, 1:1000, Abcam), β-actin (bs-0061R, 1:5000, Bioss) at 4 °C overnight.

Techniques: Infection, Western Blot, Binding Assay, Activity Assay, Luciferase, Reporter Assay, Pull Down Assay, Quantitative RT-PCR, Staining, Comparison

Journal: Cell Death & Disease

Article Title: Impaired SUMOylation of FoxA1 promotes nonalcoholic fatty liver disease through down-regulation of Sirt6

doi: 10.1038/s41419-024-07054-1

Figure Lengend Snippet: A Conserved FoxA1 SUMOylation sites at lysine residue K6. B HEK293T cells were transfected with WT-FoxA1 or FoxA1 mutants (K6R, K266R, K388R) in combination with His-Sumo2, Flag-Ubc9. Ni-NTA pull-down assay evaluated the interaction between FoxA1 and Sumo2. AML-12 cells were transfected with WT-FoxA1 or FoxA1 K6R with or without treatment with PAL. C , D RT-qPCR and Western blotting detected Sirt6 and Pparα expression. E Dual-luciferase reporter assay determined the transcription activity of Sirt6. F SUMOylation Assay Ultra Kit measured FoxA1 SUMOylation level. G The transcription activity of Sirt6 in hepatocytes with indicated treatments was measured by dual-luciferase reporter assay. Data was repeated at least 3 times. * p < 0.05, ** p < 0.01, and *** p < 0.001. For ( C – G ), One-way ANOVA followed by Tukey’s multiple comparison test was performed.

Article Snippet: The blots were blocked by skim milk and incubated using the primary antibodies against Sirt6 (A18468, 1:500, ABclonal), Cpt1a (ab234111, 1:1000, Abcam), Acox1 (ab184032, 1:1000, Abcam), Lpl (ab91606, 1:1000, Abcam), Pparα (bs-3614R, 1:500, Bioss), Cyp4a14 (ab3573, 1:1000, Abcam), FoxA1 (A15278, 1:500, ABclonal), Sae1 (ab185949, 1:1000, Abcam), Sae2 (ab185955, 1:1000, Abcam), Ubc9 (ab33044, 1:1000, Abcam), Pias1/2 (ab77231, 1:1000, Abcam), Pias3 (ab105178, 1:1000, Abcam), Pias4 (ab137500, 1:1000, Abcam), FoxO1 (ab70382, 1:2000, Abcam), Ac-FoxO1 (PA5-104560, 1:1000, Thermo Fisher), Ac-K (ab190479, 1:1000, Abcam), Cpt2 (ab181114, 1:1000, Abcam), β-actin (bs-0061R, 1:5000, Bioss) at 4 °C overnight.

Techniques: Residue, Transfection, Pull Down Assay, Quantitative RT-PCR, Western Blot, Expressing, Luciferase, Reporter Assay, Activity Assay, Comparison

Journal: Cell Death & Disease

Article Title: Impaired SUMOylation of FoxA1 promotes nonalcoholic fatty liver disease through down-regulation of Sirt6

doi: 10.1038/s41419-024-07054-1

Figure Lengend Snippet: A The primary hepatocytes were transfected with Ad-shMdm2, Ad-shMdm4, Ad-shUbox5, and FoxA1 protein levels were determined by Western blotting. B , C Co-IP confirmed the exogenous and endogenous interplay between Mdm2 and FoxA1 proteins. D The primary hepatocytes were treated with PAL together with or without MG132 (100 nM). Western blotting analysis of FoxA1 protein level. E The primary hepatocytes were infected with Ad-Mdm2 or Ad-NC, followed by treatment with PAL or/ and MG132. FoxA1 protein level was detected by Western blotting. F The primary hepatocytes were infected with Ad-shMdm2 or Ad-shNC, and then exposed to CHX for 1, 2, 4 h. Western blotting analysis of FoxA1 protein level. G AML-12 cells were transfected with WT-FoxA1 or FoxA1 K6R, and then exposed to CHX for 1, 2, 4 h. Western blotting analysis of FoxA1 protein level. H , I The ubiquitylation level of FoxA1 in primary hepatocytes and AML-12 cells was evaluated by Co-IP. J The protein levels of Mdm2, FoxA1, Sirt6 and Pparα in primary hepatocytes were determined by Western blotting. K Lipid droplet formation was observed by BODIPY staining (scale bar = 100 µm). Data was repeated at least 3 times. * p < 0.05, ** p < 0.01, and *** p < 0.001. For ( A , F , G ), Student’s t test was performed. For ( D , E , J – K ), One-way ANOVA followed by Tukey’s multiple comparison test was performed.

Article Snippet: The blots were blocked by skim milk and incubated using the primary antibodies against Sirt6 (A18468, 1:500, ABclonal), Cpt1a (ab234111, 1:1000, Abcam), Acox1 (ab184032, 1:1000, Abcam), Lpl (ab91606, 1:1000, Abcam), Pparα (bs-3614R, 1:500, Bioss), Cyp4a14 (ab3573, 1:1000, Abcam), FoxA1 (A15278, 1:500, ABclonal), Sae1 (ab185949, 1:1000, Abcam), Sae2 (ab185955, 1:1000, Abcam), Ubc9 (ab33044, 1:1000, Abcam), Pias1/2 (ab77231, 1:1000, Abcam), Pias3 (ab105178, 1:1000, Abcam), Pias4 (ab137500, 1:1000, Abcam), FoxO1 (ab70382, 1:2000, Abcam), Ac-FoxO1 (PA5-104560, 1:1000, Thermo Fisher), Ac-K (ab190479, 1:1000, Abcam), Cpt2 (ab181114, 1:1000, Abcam), β-actin (bs-0061R, 1:5000, Bioss) at 4 °C overnight.

Techniques: Transfection, Western Blot, Co-Immunoprecipitation Assay, Infection, Staining, Comparison

Journal: Cell Death & Disease

Article Title: Impaired SUMOylation of FoxA1 promotes nonalcoholic fatty liver disease through down-regulation of Sirt6

doi: 10.1038/s41419-024-07054-1

Figure Lengend Snippet: The mice were treated with GA or N106 during HFD or normal diet feeding for 12 weeks. A , B Liver weight, white adipose tissue (WAT) weight, brown adipose tissue (BAT), inguinal white adipose tissue (iWAT), and epigonadal white adipose tissue (eWAT) were measured. C Blood glucose levels of mice was detected against time after glucose or insulin injection. D – F Serum ALT, liver cholesterol and triglycerides levels were detected. G HE staining determined the pathological changes in livers (scale bar = 50 µm). H FoxA1, Sirt6, and Pparα expression in liver tissues was evaluated by immunohistochemical staining (scale bar = 50 µm). Primary hepatocytes were isolated from mice in different groups. I RT-qPCR analysis of Cd36, Fasn, Dgat, Adipoq, Fabp4, Cebpa, Pparg, Atgl, Cpt1a and Cpt2 mRNA levels in livers from mice treated with GA or N106 during HFD or normal diet feeding for 12 weeks. J BODIPY staining detected lipid droplet formation (scale bar = 100 µm). K FoxA1 SUMOylation level was measured by SUMOylation Assay Ultra Kit. ( L ) Western blotting analysis of FoxA1, Sirt6, and Pparα protein abundance. N = 6, * p < 0.05, ** p < 0.01, and *** p < 0.001. For ( C ), ANOVA for repeated measurement was performed; For ( A , B , D – F , I – L ), One-way ANOVA followed by Tukey’s multiple comparison test was performed.

Article Snippet: The blots were blocked by skim milk and incubated using the primary antibodies against Sirt6 (A18468, 1:500, ABclonal), Cpt1a (ab234111, 1:1000, Abcam), Acox1 (ab184032, 1:1000, Abcam), Lpl (ab91606, 1:1000, Abcam), Pparα (bs-3614R, 1:500, Bioss), Cyp4a14 (ab3573, 1:1000, Abcam), FoxA1 (A15278, 1:500, ABclonal), Sae1 (ab185949, 1:1000, Abcam), Sae2 (ab185955, 1:1000, Abcam), Ubc9 (ab33044, 1:1000, Abcam), Pias1/2 (ab77231, 1:1000, Abcam), Pias3 (ab105178, 1:1000, Abcam), Pias4 (ab137500, 1:1000, Abcam), FoxO1 (ab70382, 1:2000, Abcam), Ac-FoxO1 (PA5-104560, 1:1000, Thermo Fisher), Ac-K (ab190479, 1:1000, Abcam), Cpt2 (ab181114, 1:1000, Abcam), β-actin (bs-0061R, 1:5000, Bioss) at 4 °C overnight.

Techniques: Injection, Staining, Expressing, Immunohistochemical staining, Isolation, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Comparison

Journal: The FASEB Journal

Article Title: Cysteine‐rich 61 inhibition attenuates hepatic insulin resistance and improves lipid metabolism in high‐fat diet fed mice and HepG2 cells

doi: 10.1096/fj.202400860R

Figure Lengend Snippet: List of antibodies used for western blotting.

Article Snippet: Antibodies against Cyr61 (#39382), Sirt6 (#12486), phosphorylated (p)‐AMPKα (Thr172) (#2531), AMPKα (#2532), p‐insulin receptor (IR) β (Tyr1361) (#3023), IR β (#3025), insulin receptor substrate 1 (IRS‐1) (#2382), p‐ protein kinase B (Akt) (Ser473) (#9271), Akt (#9272), p‐ glycogen synthase kinase‐3 alpha/beta (GSK‐3α/β) (Ser21/9) (#8566), GSK‐3α/β (#5676), p‐ nuclear factor kappa B (NF‐κB) p65 (Ser536) (#3033), NF‐κB p65 (#4764), p‐stress‐activated protein kinase (SAPK)/ c‐Jun N‐terminal kinase (JNK) (Thr183/Tyr185) (#9251), SAPK/JNK (#9252), and α‐tubulin (#2144) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot

Journal: The FASEB Journal

Article Title: Cysteine‐rich 61 inhibition attenuates hepatic insulin resistance and improves lipid metabolism in high‐fat diet fed mice and HepG2 cells

doi: 10.1096/fj.202400860R

Figure Lengend Snippet: Primer sequences used for quantitative real time‐PCR.

Article Snippet: Antibodies against Cyr61 (#39382), Sirt6 (#12486), phosphorylated (p)‐AMPKα (Thr172) (#2531), AMPKα (#2532), p‐insulin receptor (IR) β (Tyr1361) (#3023), IR β (#3025), insulin receptor substrate 1 (IRS‐1) (#2382), p‐ protein kinase B (Akt) (Ser473) (#9271), Akt (#9272), p‐ glycogen synthase kinase‐3 alpha/beta (GSK‐3α/β) (Ser21/9) (#8566), GSK‐3α/β (#5676), p‐ nuclear factor kappa B (NF‐κB) p65 (Ser536) (#3033), NF‐κB p65 (#4764), p‐stress‐activated protein kinase (SAPK)/ c‐Jun N‐terminal kinase (JNK) (Thr183/Tyr185) (#9251), SAPK/JNK (#9252), and α‐tubulin (#2144) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques:

Journal: The FASEB Journal

Article Title: Cysteine‐rich 61 inhibition attenuates hepatic insulin resistance and improves lipid metabolism in high‐fat diet fed mice and HepG2 cells

doi: 10.1096/fj.202400860R

Figure Lengend Snippet: Hepatic Cyr61 expression is increased in HFD‐fed mice. (A) Representative images of immunohistochemical staining using anti‐Cyr61 in liver sections from CD or HFD‐fed mice. Scale bar: 50 μm. (B) Gene expression of Cyr61 in liver sections from CD or HFD‐fed mice. (C) Hepatic Cyr61 protein levels detected via western blotting in HFD and CD‐fed mice. (D) Plasma level of Cyr61 in CD or HFD‐fed mice. Data shown represent the mean ± standard error of mean. For B and D, statistical significance was determined using the Student's t ‐test. ** p < .01, *** p < .001. CD, control diet; Cyr61, cysteine‐rich 61; HFD, high‐fat diet.

Article Snippet: Antibodies against Cyr61 (#39382), Sirt6 (#12486), phosphorylated (p)‐AMPKα (Thr172) (#2531), AMPKα (#2532), p‐insulin receptor (IR) β (Tyr1361) (#3023), IR β (#3025), insulin receptor substrate 1 (IRS‐1) (#2382), p‐ protein kinase B (Akt) (Ser473) (#9271), Akt (#9272), p‐ glycogen synthase kinase‐3 alpha/beta (GSK‐3α/β) (Ser21/9) (#8566), GSK‐3α/β (#5676), p‐ nuclear factor kappa B (NF‐κB) p65 (Ser536) (#3033), NF‐κB p65 (#4764), p‐stress‐activated protein kinase (SAPK)/ c‐Jun N‐terminal kinase (JNK) (Thr183/Tyr185) (#9251), SAPK/JNK (#9252), and α‐tubulin (#2144) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Gene Expression, Western Blot, Clinical Proteomics, Control

Journal: The FASEB Journal

Article Title: Cysteine‐rich 61 inhibition attenuates hepatic insulin resistance and improves lipid metabolism in high‐fat diet fed mice and HepG2 cells

doi: 10.1096/fj.202400860R

Figure Lengend Snippet: Hepatic steatosis and inflammation are induced in Cyr61 overexpression (AAV‐Cyr61) mice. (A) Cyr61 overexpression was induced via tail vein injection of hepatocyte‐specific AAV‐Cyr61 at 6 weeks before euthanization. (B) Representative images of H&E, Oil red O, and IHC staining of liver sections from AAV‐GFP mice or AAV‐Cyr61 mice. Scale bar: 20 μm. (C) Hepatic TG content. (D) mRNA expression levels of IL‐6, TNF‐α, IL‐1β, and MCP‐1 determined via real‐time polymerase chain reaction. (E) Plasma ALT, AST, TG, and FFA levels in AAV‐GFP or AAV‐Cyr61 mice. Data shown represent the mean ± standard error of mean. Significance was determined using the Student's t ‐test. * p < .05, ** p < .01, *** p < .001. AAV, adeno‐associated virus; ALT, alanine aminotransferase; AST, aspartate transaminase; Cyr61, cysteine‐rich 61; FFA, free fatty acid; GFP, green fluorescent protein; H&E, hematoxylin and eosin; IHC, immunohistochemistry; IL‐6, interleukin 6; MCP‐1, monocyte chemoattractant protein‐1; TG, triglyceride; TNF‐α, tumor necrosis factor alpha.

Article Snippet: Antibodies against Cyr61 (#39382), Sirt6 (#12486), phosphorylated (p)‐AMPKα (Thr172) (#2531), AMPKα (#2532), p‐insulin receptor (IR) β (Tyr1361) (#3023), IR β (#3025), insulin receptor substrate 1 (IRS‐1) (#2382), p‐ protein kinase B (Akt) (Ser473) (#9271), Akt (#9272), p‐ glycogen synthase kinase‐3 alpha/beta (GSK‐3α/β) (Ser21/9) (#8566), GSK‐3α/β (#5676), p‐ nuclear factor kappa B (NF‐κB) p65 (Ser536) (#3033), NF‐κB p65 (#4764), p‐stress‐activated protein kinase (SAPK)/ c‐Jun N‐terminal kinase (JNK) (Thr183/Tyr185) (#9251), SAPK/JNK (#9252), and α‐tubulin (#2144) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Over Expression, Injection, Immunohistochemistry, Expressing, Real-time Polymerase Chain Reaction, Clinical Proteomics, Virus

Journal: The FASEB Journal

Article Title: Cysteine‐rich 61 inhibition attenuates hepatic insulin resistance and improves lipid metabolism in high‐fat diet fed mice and HepG2 cells

doi: 10.1096/fj.202400860R

Figure Lengend Snippet: Hepatic lipid accumulation and insulin resistance are induced in Cyr61 overexpression (AAV‐Cyr61) mice. Blood glucose levels in AAV‐GFP or AAV‐Cyr61 during (A) ITT and (B) GTT assays after 6 h of fasting. Relative mRNA expression levels of key genes involved in (C) lipogenesis and (D) fatty acid oxidation in the liver of AAV‐GFP mice or AAV‐Cyr61 mice. (E) Quantitative analysis of Sirtuin genes determined by quantitative polymerase chain reaction. Data shown represent the mean ± standard error of mean. Statistical significance was determined using the t ‐test. * p < .05, ** p < .01, *** p < .001. AAV, adeno‐associated virus; AUC, area under the curve; Cyr61, cysteine‐rich 61; GTT, glucose tolerance test; ITT, insulin tolerance test.

Article Snippet: Antibodies against Cyr61 (#39382), Sirt6 (#12486), phosphorylated (p)‐AMPKα (Thr172) (#2531), AMPKα (#2532), p‐insulin receptor (IR) β (Tyr1361) (#3023), IR β (#3025), insulin receptor substrate 1 (IRS‐1) (#2382), p‐ protein kinase B (Akt) (Ser473) (#9271), Akt (#9272), p‐ glycogen synthase kinase‐3 alpha/beta (GSK‐3α/β) (Ser21/9) (#8566), GSK‐3α/β (#5676), p‐ nuclear factor kappa B (NF‐κB) p65 (Ser536) (#3033), NF‐κB p65 (#4764), p‐stress‐activated protein kinase (SAPK)/ c‐Jun N‐terminal kinase (JNK) (Thr183/Tyr185) (#9251), SAPK/JNK (#9252), and α‐tubulin (#2144) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Virus

Journal: The FASEB Journal

Article Title: Cysteine‐rich 61 inhibition attenuates hepatic insulin resistance and improves lipid metabolism in high‐fat diet fed mice and HepG2 cells

doi: 10.1096/fj.202400860R

Figure Lengend Snippet: Hepatic inflammation and liver steatosis are ameliorated after Cyr61 knockdown (AAV‐shCyr61) mice. (A) Male C57BL/6J mice were fed CD ( n = 7) or HFD ( n = 7) for 18 weeks. AAV‐shRNA (2 × 10 11 viral particles/mouse) were injected intravenously at 6 weeks before termination. (B) mRNA expression of Cyr61 in the liver of mice with AAV‐GFP or AAV‐shCyr61 transfection. (C) Representative images of H&E and Oil Red O staining in mouse liver sections. Scale bar: 50 μm. (D) Hepatic TG content. (E) Plasma ALT, AST, and TG levels in AAV‐GFP or AAV‐shCyr61. (F) mRNA levels of pro‐inflammatory cytokines (IL‐6, TNF‐α, IL‐1β, and MCP‐1) in AAV‐GFP or AAV‐shCyr61 mice. Data shown represent the mean ± standard error of mean. Statistical significance was determined using the two‐way ANOVA. ** p < .01, *** p < .001 vs CD/AAV‐GFP. # p < .05, ## p < .01, ### p < .001 vs HFD/AAV‐GFP. AAV, adeno‐associated virus; ALT, alanine aminotransferase; AST, aspartate transaminase; CD, control diet; Cyr61, cysteine‐rich 61; FFA, free fatty acid ; GFP, green fluorescent protein; H&E, hematoxylin and eosin; HFD, high‐fat diet; IHC, immunohistochemistry; IL‐6, interleukin 6; MCP‐1, monocyte chemoattractant protein‐1; shRNA, short hairpin RNA; TG, triglyceride; TNF‐α, tumor necrosis factor alpha.

Article Snippet: Antibodies against Cyr61 (#39382), Sirt6 (#12486), phosphorylated (p)‐AMPKα (Thr172) (#2531), AMPKα (#2532), p‐insulin receptor (IR) β (Tyr1361) (#3023), IR β (#3025), insulin receptor substrate 1 (IRS‐1) (#2382), p‐ protein kinase B (Akt) (Ser473) (#9271), Akt (#9272), p‐ glycogen synthase kinase‐3 alpha/beta (GSK‐3α/β) (Ser21/9) (#8566), GSK‐3α/β (#5676), p‐ nuclear factor kappa B (NF‐κB) p65 (Ser536) (#3033), NF‐κB p65 (#4764), p‐stress‐activated protein kinase (SAPK)/ c‐Jun N‐terminal kinase (JNK) (Thr183/Tyr185) (#9251), SAPK/JNK (#9252), and α‐tubulin (#2144) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Knockdown, shRNA, Injection, Expressing, Transfection, Staining, Clinical Proteomics, Virus, Control, Immunohistochemistry

Journal: The FASEB Journal

Article Title: Cysteine‐rich 61 inhibition attenuates hepatic insulin resistance and improves lipid metabolism in high‐fat diet fed mice and HepG2 cells

doi: 10.1096/fj.202400860R

Figure Lengend Snippet: HFD‐induced insulin resistance and altered lipid metabolism‐related genes are improved in Cyr61 knockdown (AAV‐shCyr61) mice. Blood glucose levels in AAV‐shGFP or AAV‐shCyr61 during (A) GTT and (B) ITT assays after 6 h of fasting. Relative mRNA expression levels of key genes involved in (C) lipogenesis and (D) fatty acid oxidation in the liver of AAV‐shGFP or AAV‐shCyr61 mice. (E) Representative hepatic expression of Sirt6, p‐AMPK, and T‐AMPK were determined using western blotting. (F) Mice were intraperitoneally injected with insulin after 6 h of fasting. Representative hepatic expression of IRS1 and AKT was determined using western blotting. Statistical significance was determined using the two‐way ANOVA. * p < .05, ** p < .01, *** p < .001 vs CD/AAV‐GFP. # p < .05, ## p < .01, ### p < .001 vs HFD/AAV‐GFP. AAV, adeno‐associated virus; AKT, protein kinase B; AUC, area under the curve; Cyr61, cysteine‐rich 61; GTT, glucose tolerance test; IRS1, insulin receptor substrate 1; ITT, insulin tolerance test; p‐AMPK, phosphorylated AMP‐activated protein kinase; Sirt6, sirtuin 6.

Article Snippet: Antibodies against Cyr61 (#39382), Sirt6 (#12486), phosphorylated (p)‐AMPKα (Thr172) (#2531), AMPKα (#2532), p‐insulin receptor (IR) β (Tyr1361) (#3023), IR β (#3025), insulin receptor substrate 1 (IRS‐1) (#2382), p‐ protein kinase B (Akt) (Ser473) (#9271), Akt (#9272), p‐ glycogen synthase kinase‐3 alpha/beta (GSK‐3α/β) (Ser21/9) (#8566), GSK‐3α/β (#5676), p‐ nuclear factor kappa B (NF‐κB) p65 (Ser536) (#3033), NF‐κB p65 (#4764), p‐stress‐activated protein kinase (SAPK)/ c‐Jun N‐terminal kinase (JNK) (Thr183/Tyr185) (#9251), SAPK/JNK (#9252), and α‐tubulin (#2144) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Knockdown, Expressing, Western Blot, Injection, Virus

Journal: The FASEB Journal

Article Title: Cysteine‐rich 61 inhibition attenuates hepatic insulin resistance and improves lipid metabolism in high‐fat diet fed mice and HepG2 cells

doi: 10.1096/fj.202400860R

Figure Lengend Snippet: PA treatment increased Cyr61 expression in HepG2 cells. (A) HepG2 cells were incubated with the indicated dosage of PA for 12 h, after which Cyr61 expression was measured using real‐time PCR. (B) The level of Cyr61 was determined in cell culture media of HepG2 cells treated with PA. (C) HepG2 cells were incubated with PA for 12 h, after which inflammatory cytokine gene expression levels were measured using real‐time PCR. (D) The expression levels of the inflammatory protein markers p‐NF‐κB and p‐JNK were measured using western blotting. (E) After treatment with PA, insulin resistance in HepG2 cells was determined by measuring p‐AKT and p‐GSK3 α/β levels via western blotting. Data shown represent the mean ± standard error of mean. For A and C, statistical significance was determined using the one‐way ANOVA. * p < .05, ** p < .01, *** p < .001 vs BSA. BSA, bovine serum albumin; Cyr61, cysteine‐rich 61; PA, palmitic acid; p‐AKT, phosphorylated protein kinase B; PCR, polymerase chain reaction; p‐GSK3 α/β, phosphorylated glycogen synthase kinase‐3 alpha/beta; p‐JNK, phosphorylated c‐Jun N‐terminal kinase; p‐NF‐κB, phosphorylated nuclear factor kappa B.

Article Snippet: Antibodies against Cyr61 (#39382), Sirt6 (#12486), phosphorylated (p)‐AMPKα (Thr172) (#2531), AMPKα (#2532), p‐insulin receptor (IR) β (Tyr1361) (#3023), IR β (#3025), insulin receptor substrate 1 (IRS‐1) (#2382), p‐ protein kinase B (Akt) (Ser473) (#9271), Akt (#9272), p‐ glycogen synthase kinase‐3 alpha/beta (GSK‐3α/β) (Ser21/9) (#8566), GSK‐3α/β (#5676), p‐ nuclear factor kappa B (NF‐κB) p65 (Ser536) (#3033), NF‐κB p65 (#4764), p‐stress‐activated protein kinase (SAPK)/ c‐Jun N‐terminal kinase (JNK) (Thr183/Tyr185) (#9251), SAPK/JNK (#9252), and α‐tubulin (#2144) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Cell Culture, Gene Expression, Western Blot, Polymerase Chain Reaction

Journal: The FASEB Journal

Article Title: Cysteine‐rich 61 inhibition attenuates hepatic insulin resistance and improves lipid metabolism in high‐fat diet fed mice and HepG2 cells

doi: 10.1096/fj.202400860R

Figure Lengend Snippet: Cyr61 knockdown inhibits inflammation and restores insulin resistance in PA‐treated HepG2 cells. HepG2 cells were transfected with siRNA of GFP or Cyr61. siGFP‐ or si Cyr61‐transfected HepG2 cells were treated with 0.4 mM palmitate. (A) Quantitative analysis of Cyr61 expression by quantitative PCR. (B) Expression levels of inflammatory genes (IL‐6, IL‐1β, and TNF‐α) were determined by real‐time PCR. (C) Relative mRNA expression level of key genes involved in lipogenesis and fatty acid oxidation. (D) Levels of p‐NF‐κB and p‐JNK were determined by immunoblotting. (E) The levels of p‐AKT and p‐GSKα/β were determined by immunoblotting. Data shown represent the mean ± standard error of mean. For A, statistical significance was determined using the Student's t ‐test. *** p < .001. For B and C, significance was determined using the two‐way ANOVA. * p < .05, ** p < .01, *** p < .001 vs BSA/siGFP. # p < .05, ## p < .01 vs PA/siGFP. Cyr61, cysteine‐rich 61; GFP, green fluorescent protein; IL‐6, interleukin 6; NF‐κB, phosphorylated nuclear factor kappa B; p‐AKT, phosphorylated protein kinase B; PCR, polymerase chain reaction; p‐GSK3 α/β, phosphorylated glycogen synthase kinase‐3 alpha/beta; p‐JNK, phosphorylated c‐Jun N‐terminal kinase; siRNA, small‐interfering RNA; TNF‐α, tumor necrosis factor alpha.

Article Snippet: Antibodies against Cyr61 (#39382), Sirt6 (#12486), phosphorylated (p)‐AMPKα (Thr172) (#2531), AMPKα (#2532), p‐insulin receptor (IR) β (Tyr1361) (#3023), IR β (#3025), insulin receptor substrate 1 (IRS‐1) (#2382), p‐ protein kinase B (Akt) (Ser473) (#9271), Akt (#9272), p‐ glycogen synthase kinase‐3 alpha/beta (GSK‐3α/β) (Ser21/9) (#8566), GSK‐3α/β (#5676), p‐ nuclear factor kappa B (NF‐κB) p65 (Ser536) (#3033), NF‐κB p65 (#4764), p‐stress‐activated protein kinase (SAPK)/ c‐Jun N‐terminal kinase (JNK) (Thr183/Tyr185) (#9251), SAPK/JNK (#9252), and α‐tubulin (#2144) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Knockdown, Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction, Small Interfering RNA

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Impaired Reciprocal Regulation between SIRT6 and TGF-β Signaling in Fatty Liver

doi: 10.1096/fj.202101518R

Figure Lengend Snippet: (A) Schematic of the human SIRT6 promoter with potential SBEs and CTCF binding sites. (B) Representative SMAD3 and CTCF binding peaks at the SIRT6 promoter and enhancer regions from one of three ChIP-Seq experiments in HepG2 cells with or without TGF-β treatment. The region of chromosome 19 (chr19) is indicated. (C) SMAD3 binding to SBE1 and SBE2 from ChIP-qPCR experiments is presented as the fold recruitment. Data are presented as the mean ± SEM of three independent experiments. *, p < 0.05; compared with IgG group. (D) Relative mRNA levels of SIRT6 and SERPINE1 were determined in MEFs exposed to 200 pM TGF-β for 24 h. *, p < 0.05; compared to WT. (E) SIRT6 was detected by Western blotting in MEFs with different genotyping (left) and HepG2 stable cell lines with or without SMAD3 knockdown (right). GAPDH or ACTIN served as the loading control.

Article Snippet: 20 The antibody against SIRT6 is from NOVUS (NB100–2522).

Techniques: Binding Assay, ChIP-sequencing, ChIP-qPCR, Western Blot, Stable Transfection, Knockdown, Control

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Impaired Reciprocal Regulation between SIRT6 and TGF-β Signaling in Fatty Liver

doi: 10.1096/fj.202101518R

Figure Lengend Snippet: (A) Relative mRNA levels of the indicated TGF-β target genes in HepG2 cells transfected with empty vector (EV) or Flag-SIRT6 and exposed to 200 pM TGF-β for 24 h. Data are presented as mean ± SEM relative to the amount in EV-transfected cells exposed to TGF-β treatment (n = 3 – 4). *, p < 0.05; compared with EV group with TGF-β treatment. (B) Co-immunoprecipitation of SMAD3, SPTBN1, and Flag-SIRT6 was performed in HepG2 cells with or without exposure to 200 pM TGF-β for 3 h, using an irrelevant IgG antibody or anti-FLAG antibody. IP, immunoprecipitation. Representative data from 1 of 3 independent experiments are shown. (C) The effect of SIRT6 overexpression on SPTBN1 acetylation in Huh7 cells was determined by immunoprecipitating SPTBN1 and Western blotting for the presence of acetylation with SPTBN1. Representative data from 1 of 3 independent experiments are shown. (D) The effect of SIRT6 overexpression on SPTBN1, SMAD3, Pre-SREBP1, n-SREBP1, and LDLR abundance in HepG2 cells and Huh7 cells was determined by Western blotting. VINCULIN served as the loading control. EV, empty vector. Representative data from 1 of 3 independent experiments are shown. (E) Relative mRNA levels of SMAD3 and SPTBN1 were determined in SNU398 cells transfected with empty vector (EV) or Flag-SIRT6. Data are presented as mean ± SEM relative to the amount in EV-transfected cells (n = 3). No significant differences were detected with t-test, compared to EV group.

Article Snippet: 20 The antibody against SIRT6 is from NOVUS (NB100–2522).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Over Expression, Western Blot, Control

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Impaired Reciprocal Regulation between SIRT6 and TGF-β Signaling in Fatty Liver

doi: 10.1096/fj.202101518R

Figure Lengend Snippet: WT or SPTBN1+/− mice (10 – 12 weeks old) were fed a normal chow (NC) diet or a HFD for 12 – 16 weeks. (A) Body weight and serum cholesterol and serum triglyceride concentrations are shown for mice after receiving a HFD for 12 – 16 weeks. Data are presented as mean ± SEM of 3 – 4 mice per group. *, p < 0.05 compared to WT. (B) Liver histology and SIRT6 immunohistochemistry (IHC) results are shown for mice after 12–16 weeks on the indicated diets. Hematoxylin & eosin (H&E) and IHC images are shown at two magnifications: 10×, scale bar = 100 μm; 20×, scale bar = 50 μm. C, central vein; P, portal tracts; arrows indicate inflammation. Representative zones between central vein and portal tracts are marked by the black dashed line:1, indicates area close to central vein; 2, indicates area in the middle; 3, indicates area close to portal tracts. (C) SIRT6 abundance was quantified from SIRT6 IHC in liver tissue from mice of the indicated genotypes (n = 3). **, p < 0.01; compared with WT group on the same diet; ##, p < 0.01, compared with WT group on NC diet. (D) Profiles of SIRT6 transcripts in the regions of the liver from the central vein to the portal node in human (left) and mouse (right). Each profile is normalized to the maximal expression across zones. The line indicates the mean, and the shaded region indicates the SEM.

Article Snippet: 20 The antibody against SIRT6 is from NOVUS (NB100–2522).

Techniques: Immunohistochemistry, Expressing

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Impaired Reciprocal Regulation between SIRT6 and TGF-β Signaling in Fatty Liver

doi: 10.1096/fj.202101518R

Figure Lengend Snippet: MEFs were transfected with empty vector (EV) or FLAG-SIRT6 and exposed to vehicle (Veh) or stressed with TNF-α (20 ng/ml) and cycloheximide (CHX, 10 μg/ml) for 3 h. (A) In addition to the EV or FLAG-SIRT6, MEFs were transfected with plasmids encoding Renilla and luciferase controlled by the indicated promoter. Luciferase activity from the LDLR promoter or the SCD1 promoter was normalized using the activity of Renilla luciferase and the mean ± SEM relative to the vehicle-treated WT were plotted (n = 3 – 4 independent experiments). (B) Transcript abundance of the indicated SREBP target genes is presented relative to that in WT MEFs transfected with empty vector and exposed to vehicle. Data are presented as the mean ± SEM (n = 3). In A and B, *, p < 0.05, compared with vehicle-treated cells; #, p < 0.05, stressed SPTBN1+/− EV versus FLAG-SIRT6.

Article Snippet: 20 The antibody against SIRT6 is from NOVUS (NB100–2522).

Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay