aav  (Vector Biolabs)

Journal: Cell Death & Disease

Article Title: Adipocyte-expressed SIRT3 manipulates carnitine pool to orchestrate metabolic reprogramming and polarization of macrophages

doi: 10.1038/s41419-025-07699-6

Figure Lengend Snippet: The mRNA expression of Tnf-α ( A ) Il-6 ( B ), Il-1β ( C ) and iNos ( D ) in BMDMs. The mRNA expression of Mrc1 ( E ) Mgl2 ( F ) Arg1 ( G ) and Chil3 ( H ) in BMDMs. n = 6 independent experiments. I The F4/80/CD11c double-positive and F4/80/CD206 double-positive macrophages were analyzed by flow cytometry. n = 5 independent experiments. J The mRNA expression of Mcp-1 , Mip-1α , Ccl5 , Ccl11 , Cxcl10 and Cxcl11 in BMDMs. n = 6 independent experiments. K Transwell migration of macrophages towards CM from vector and SIRT3OE adipocytes. n = 6 independent experiments. Data are expressed as means ± SEM. # P < 0.05, ## p < 0.01, ### p < 0.001, vector vs. control; * P < 0.05, ** P < 0.01, vector vs. SIRT3OE.

Article Snippet: Recombinant adeno-associated serotype 9 viruses with Ap2 promoter for SIRT3 overexpression in adipocytes (AAV- Ap2 -SIRT3) or the empty vector (AAV- Ap2 ) were acquired from GeneChem Co, Ltd (Shanghai, China).

Techniques: Expressing, Flow Cytometry, Migration, Plasmid Preparation, Control

Journal: Cell Death & Disease

Article Title: Adipocyte-expressed SIRT3 manipulates carnitine pool to orchestrate metabolic reprogramming and polarization of macrophages

doi: 10.1038/s41419-025-07699-6

Figure Lengend Snippet: A VIP of significantly altered metabolites. B Scatter diagram of PC, OC, HC, Pro-LC, Isobu-LC and LC, each dot shows a technical replicate. C Volcano plot of significantly altered metabolites between the vector and SIRT3OE cells. n = 6 replicates. D Contents of PC and LC in CM of vector and SIRT3OE adipocytes. n = 6 replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, vector vs. SIRT3OE. E Contents of PC and LC in serum of mice. n = 4 mice per group. Data are expressed as means ± SEM. ## P < 0.01, ### P < 0.001, HFD-AT-NC vs. RD-AT-NC; * P < 0.05, ** P < 0.01, *** P < 0.001, HFD-AT-NC vs. HFD - AT-SIRT3OE.

Article Snippet: Recombinant adeno-associated serotype 9 viruses with Ap2 promoter for SIRT3 overexpression in adipocytes (AAV- Ap2 -SIRT3) or the empty vector (AAV- Ap2 ) were acquired from GeneChem Co, Ltd (Shanghai, China).

Techniques: Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Adipocyte-expressed SIRT3 manipulates carnitine pool to orchestrate metabolic reprogramming and polarization of macrophages

doi: 10.1038/s41419-025-07699-6

Figure Lengend Snippet: A The acetylated CPT2 levels in vector and SIRT3OE adipocytes. n = 3 independent experiments. B The CPT2 activity in vector and SIRT3OE adipocytes. n = 6 replicates. C The CPT2 activity in Scramble and SIRT3KD adipocytes. * P < 0.05, Scramble vs. SIRT3KD. n = 3 replicates. D The substrate dependency in vector and SIRT3OE adipocytes was determined by Seahorse XF mitochondrial fuel flex test. n = 3 replicates. E The CPT2 activity was evaluated. n = 6 replicates. The contents of PC ( F ) and LC ( G ) in CM of adipocytes. n = 3 replicates. The mRNA expression of Tnf-α ( H ), Il-6 ( I ), Il-1β ( J ), iNos ( K ) and Mcp-1 ( L ) in BMDMs treated with CM of adipocytes. n = 6 replicates. M The protein expression of p-IKKα/β, IKKα, IKKβ, p-IκBα, IκBα, p-p65 and p65. β -actin was used as a loading control. n = 3 replicates. Data are expressed as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, vector vs. SIRT3OE; # P < 0.05, ## P < 0.01, ### P < 0.001, vector vs. CPT2KD; & P < 0.05, & & P < 0.01, control vs vector; $ P < 0.05, $$$ P < 0.001, CPT2KD vs. SIRT3OE-CPT2KD.

Article Snippet: Recombinant adeno-associated serotype 9 viruses with Ap2 promoter for SIRT3 overexpression in adipocytes (AAV- Ap2 -SIRT3) or the empty vector (AAV- Ap2 ) were acquired from GeneChem Co, Ltd (Shanghai, China).

Techniques: Plasmid Preparation, Activity Assay, Expressing, Control

Journal: Antioxidants

Article Title: SIRT3 Modulates Endothelial Mitochondrial Redox State during Insulin Resistance

doi: 10.3390/antiox11081611

Figure Lengend Snippet: Effects of PA on IR and mitochondrial status. Immunoblotting analysis of ( A ) phospho-IRS1, ( B ) phospho-Akt/Akt, and ( C ) phospho-GSK-3β. ( D ) Cytokine content and ( E – G ) representative images by fluorescence microscopy and FACS analysis of mitochondrial ROS detection expressed as fluorescence median ± SD ( n = 3). Scale bars = 100 µm. ( H ) Acetylated-SOD2/SOD2 ratio. ( I , J ) SIRT2, ( K , L ) SIRT3, ( M , N ) SIRT4, and ( O , P ) SIRT5 mRNA and protein levels in EC exposed to 0.5 mM PA for 48 h or treated with the corresponding highest volume of HBSS-10 mM Hepes (Ctr). Western blotting results ( n = 5) are expressed as arbitrary units (AU), while mRNA levels ( n = 3) are reported as floating bars with a line representing the median ± SD. * p < 0.05 vs. Ctr; † p < 0.01 vs. Ctr; ‡ p < 0.001 vs. Ctr.

Article Snippet: The next day, the 70–80% subconfluent cultures were transfected with 20 nM SIRT3 AAV Vector for specific human Sirtuin 3 (438081010110, Applied Biological Materials, Inc. Richmond, BC, Canada) or miRNA mimic Negative Control (MCH00000, Applied Biological Materials, Inc. Richmond, BC, Canada), in serum- and antibiotic-free medium, using Lullaby (LL70500, OZ Biosciences, Marseille, France) as transfection reagent.

Techniques: Western Blot, Fluorescence, Microscopy

Journal: Antioxidants

Article Title: SIRT3 Modulates Endothelial Mitochondrial Redox State during Insulin Resistance

doi: 10.3390/antiox11081611

Figure Lengend Snippet: SIRT3 + reduced the PA-related cytotoxicity. ( A ) Immunoblotting analysis of SIRT3 protein levels, ( B ) LDH assay cytotoxicity, ( C ) MDA, and ( D ) extracellular ROS content in EC treated with the empty transfection reagent (Lullaby) or transfected with mimic Negative Control (mimic NC), SIRT3 mimic (SIRT3 + ), mimic Negative Control and then exposed to 0.5 mM PA for 48 h (mimic NC+PA) or SIRT3 mimic before 48 h treatment with PA (SIRT3 + +PA). Control cells (Ctr) were treated with the corresponding volume of HBSS-10 mM Hepes. Western blotting results ( n = 5) are expressed as arbitrary units (AU) and represented as boxplots. ‡ p < 0.001 vs. mimic NC; § p < 0.05 vs. mimic NC+PA; # p < 0.01 vs. mimic NC+PA.

Article Snippet: The next day, the 70–80% subconfluent cultures were transfected with 20 nM SIRT3 AAV Vector for specific human Sirtuin 3 (438081010110, Applied Biological Materials, Inc. Richmond, BC, Canada) or miRNA mimic Negative Control (MCH00000, Applied Biological Materials, Inc. Richmond, BC, Canada), in serum- and antibiotic-free medium, using Lullaby (LL70500, OZ Biosciences, Marseille, France) as transfection reagent.

Techniques: Western Blot, Lactate Dehydrogenase Assay, Transfection, Negative Control

Journal: Antioxidants

Article Title: SIRT3 Modulates Endothelial Mitochondrial Redox State during Insulin Resistance

doi: 10.3390/antiox11081611

Figure Lengend Snippet: SIRT3 + decreased the PA-induced IR and oxidative stress. ( A ) phospho-IRS1, ( B ) phospho-Akt/Akt, and ( C ) phospho-GSK-3β protein expression. Representative images by fluorescence microscopy and FACS analysis of ( D – F ) intracellular and ( G – I ) mitochondrial ROS detection, expressed as MFI, and ( J ) acetylated-SOD2/SOD2 protein levels in EC transfected with mimic Negative Control (mimic NC), SIRT3 mimic (SIRT3 + ), mimic Negative Control and then exposed to 0.5 mM PA for 48 h (mimic NC+PA) or SIRT3 mimic before 48 h treatment with PA (SIRT3 + +PA). Scale bars = 100 µm. Western blotting results ( n = 5) are expressed as arbitrary units (AU) and represented as boxplots. † p < 0.01 vs. mimic NC; ‡ p < 0.001 vs. mimic NC; § p < 0.05 vs. mimic NC+PA; # p < 0.01 vs. mimic NC+PA.

Article Snippet: The next day, the 70–80% subconfluent cultures were transfected with 20 nM SIRT3 AAV Vector for specific human Sirtuin 3 (438081010110, Applied Biological Materials, Inc. Richmond, BC, Canada) or miRNA mimic Negative Control (MCH00000, Applied Biological Materials, Inc. Richmond, BC, Canada), in serum- and antibiotic-free medium, using Lullaby (LL70500, OZ Biosciences, Marseille, France) as transfection reagent.

Techniques: Expressing, Fluorescence, Microscopy, Transfection, Negative Control, Western Blot

Journal: Antioxidants

Article Title: SIRT3 Modulates Endothelial Mitochondrial Redox State during Insulin Resistance

doi: 10.3390/antiox11081611

Figure Lengend Snippet: SIRT3 + counteracted the PA-induced inflammation. ( A ) Cytokine content and protein level of ( B ) TNF-α, and ( C ) IL-6. ( D ) Representative images of confocal laser scanning microscopy (scale bars = 10 µm). ( E ) Fluorescence intensity and ( F ) protein expression levels of NF-κB in EC transfected with mimic Negative Control (mimic NC), SIRT3 mimic (SIRT3 + ), mimic Negative Control and then exposed to 0.5 mM PA for 48 h (mimic NC+PA) or SIRT3 mimic before 48 h treatment with PA (SIRT3 + +PA). Fluorescence analysis is reported as boxplots of arbitrary fluorescence units (AFU) of 5 independent experiments. Densitometric immunoblotting study ( n = 5) is expressed as arbitrary units (AU). † p < 0.01 vs. mimic NC; ‡ p < 0.001 vs. mimic NC; § p < 0.05 vs. mimic NC+PA; # p < 0.01 vs. mimic NC+PA.

Article Snippet: The next day, the 70–80% subconfluent cultures were transfected with 20 nM SIRT3 AAV Vector for specific human Sirtuin 3 (438081010110, Applied Biological Materials, Inc. Richmond, BC, Canada) or miRNA mimic Negative Control (MCH00000, Applied Biological Materials, Inc. Richmond, BC, Canada), in serum- and antibiotic-free medium, using Lullaby (LL70500, OZ Biosciences, Marseille, France) as transfection reagent.

Techniques: Confocal Laser Scanning Microscopy, Fluorescence, Expressing, Transfection, Negative Control, Western Blot

Journal: Antioxidants

Article Title: SIRT3 Modulates Endothelial Mitochondrial Redox State during Insulin Resistance

doi: 10.3390/antiox11081611

Figure Lengend Snippet: SIRT3 + reduced the PA-mediated pyroptosis. ( A ) Representative images and ( B , C ) cytometer analysis, expressed as green fluorescence median, of pyroptosis (scale bars = 100 µm). ( D ) Cytokine levels and immunoblotting analysis of ( E ) IL-1β, ( F ) IL-18, ( G ) NLRP3, ( H ) ASC, and ( I ) caspase-1. ( J ) Fluorescence intensity and ( K ) representative images of confocal laser scanning microscopy (scale bars = 10 µm) of caspase-1 in EC transfected with mimic Negative Control (mimic NC), SIRT3 mimic (SIRT3 + ), mimic Negative Control and then exposed to 0.5 mM PA for 48 h (mimic NC+PA) or SIRT3 mimic before 48 h treatment with PA (SIRT3 + +PA). Fluorescence analysis ( n = 5) is reported as boxplots of arbitrary fluorescence units (AFU), protein expression levels ( n = 5) expressed as arbitrary units (AU). † p < 0.01 vs. mimic NC; ‡ p < 0.001 mimic NC; § p < 0.05 vs. mimic NC+PA; # p < 0.01 vs. mimic NC+PA.

Article Snippet: The next day, the 70–80% subconfluent cultures were transfected with 20 nM SIRT3 AAV Vector for specific human Sirtuin 3 (438081010110, Applied Biological Materials, Inc. Richmond, BC, Canada) or miRNA mimic Negative Control (MCH00000, Applied Biological Materials, Inc. Richmond, BC, Canada), in serum- and antibiotic-free medium, using Lullaby (LL70500, OZ Biosciences, Marseille, France) as transfection reagent.

Techniques: Cytometry, Fluorescence, Western Blot, Confocal Laser Scanning Microscopy, Transfection, Negative Control, Expressing

Journal: Antioxidants

Article Title: SIRT3 Modulates Endothelial Mitochondrial Redox State during Insulin Resistance

doi: 10.3390/antiox11081611

Figure Lengend Snippet: SIRT3 + decreased the PA-induced autophagy. Representative images of fluorescence microscopy and flow cytometry analysis of ( A – C ) green detection reagent and ( F – H ) Lysotracker Red, quantified as MFI (scale bars = 100 µm). Western blotting analysis of ( D ) beclin-1, ( E ) p62, ( I ) ATG5, and ( J ) LC3B II/I in EC transfected with mimic Negative Control (mimic NC), SIRT3 mimic (SIRT3 + ), mimic Negative Control and then exposed to 0.5 mM PA for 48 h (mimic NC+PA) or SIRT3 mimic before 48 h treatment with PA (SIRT3 + +PA). Western blotting results ( n = 5) are expressed as arbitrary units (AU) and represented as boxplots. † p < 0.01 vs. mimic NC; ‡ p < 0.001 vs. mimic NC; § p < 0.05 vs. mimic NC+PA; # p < 0.01 vs. mimic NC+PA.

Article Snippet: The next day, the 70–80% subconfluent cultures were transfected with 20 nM SIRT3 AAV Vector for specific human Sirtuin 3 (438081010110, Applied Biological Materials, Inc. Richmond, BC, Canada) or miRNA mimic Negative Control (MCH00000, Applied Biological Materials, Inc. Richmond, BC, Canada), in serum- and antibiotic-free medium, using Lullaby (LL70500, OZ Biosciences, Marseille, France) as transfection reagent.

Techniques: Fluorescence, Microscopy, Flow Cytometry, Western Blot, Transfection, Negative Control

Journal: Antioxidants

Article Title: SIRT3 Modulates Endothelial Mitochondrial Redox State during Insulin Resistance

doi: 10.3390/antiox11081611

Figure Lengend Snippet: δVB reduced the PA-induced mitochondrial dysfunction. ( A ) Representative images by fluorescence microscopy (scale bars = 100 µm) and ( B , C ) FACS analysis of mitochondrial ROS detection (MFI). Levels of mRNA and immunoblotting analysis of ( D , E ) SIRT2, ( F , G ) SIRT3, ( H , I ) SIRT4, and ( J , K ) SIRT5 in EC exposed for 48 h to 0.5 mM δVB, 0.5 mM PA (PA), or pretreated for 16 h with δVB before 48 h PA treatment (δVB+PA). Control cells (Ctr) were treated with the corresponding highest volume of HBSS-10 mM Hepes. Western blotting results ( n = 5) are expressed as arbitrary units (AU). mRNA levels are reported as floating bars with line representing the median ± SD ( n = 3). * p < 0.05 vs. Ctr; † p < 0.01 vs. Ctr; ‡ p < 0.001 vs. Ctr; § p < 0.05 vs. PA; # p < 0.01 vs. PA.

Article Snippet: The next day, the 70–80% subconfluent cultures were transfected with 20 nM SIRT3 AAV Vector for specific human Sirtuin 3 (438081010110, Applied Biological Materials, Inc. Richmond, BC, Canada) or miRNA mimic Negative Control (MCH00000, Applied Biological Materials, Inc. Richmond, BC, Canada), in serum- and antibiotic-free medium, using Lullaby (LL70500, OZ Biosciences, Marseille, France) as transfection reagent.

Techniques: Fluorescence, Microscopy, Western Blot