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Novus Biologicals mouse anti sirt3
Mouse Anti Sirt3, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse anti sirt3 - by Bioz Stars, 2026-06

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Schematic of the mechanism by which curcumin mitigates diabetic osteoporosis. Curcumin suppresses mitochondrial oxidative stress in osteoblasts induced by a high-glucose microenvironment via activation of the Sirt3/FoxO3a signaling pathway, thereby enhancing cell viability and promoting osteogenic differentiation and extracellular matrix secretion.

Journal: Scientific Reports

Article Title: Curcumin preserves bone health compromised by diabetes by inhibiting osteoporosis through regulation of the SIRT3/FoxO3a signalling pathway

doi: 10.1038/s41598-025-15165-8

Figure Lengend Snippet: Schematic of the mechanism by which curcumin mitigates diabetic osteoporosis. Curcumin suppresses mitochondrial oxidative stress in osteoblasts induced by a high-glucose microenvironment via activation of the Sirt3/FoxO3a signaling pathway, thereby enhancing cell viability and promoting osteogenic differentiation and extracellular matrix secretion.

Article Snippet: Curcumin (Solarbio), mouse anti-SIRT3 antibody (Proteintech) , mouse anti-FoxO3a antibody (Proteintech), mouse anti-cytochrome c antibody (Proteintech), mouse anti-NRF2 antibody (Proteintech), mouse anti-NOX2 antibody (Proteintech), mouse anti-caspase 3 antibody (Proteintech), mouse anti-SOD2 antibody (Proteintech), rabbit anti-mouse SIRT3 antibody (Proteintech) Proteintech), rabbit anti-mouse Bcl2 antibody (Proteintech), mouse anti-β-actin antibody (Beijing Zhongyi Jinqiao Company), sodium pentobarbital (Sigma), paraformaldehyde (Xi’an Reagent Factory), α-D-glucose (Sigma), anti-osteocalcin (OCN) antibody (Merck & Millipore ), NQO1 (Proteintech), mouse osteoprotegerin (OPG) antibody (R&D), minimum essential medium alpha (α-MEM) (Bioind), dimethyl sulfoxide Hybri-Max (Sigma), Flow Cytometry Kit (Jiangsu Biyuntian), osteogenic induction complete medium (Pythonbio), Mitochondrial Membrane Potential Assay Kit (Jiangsu Biyuntian), ROS Reactive Oxygen Species Assay Kit (Jiangsu Biyuntian).

Techniques: Activation Assay

Curcumin inhibits high glucose-induced oxidative stress and activates the SIRT3/FoxO3a signaling pathway in MC3T3-E1 cells. ( A ) DCFH-DA staining assay for ROS levels in MC3T3-E1 cells after 48 h of treatment with high glucose (HG, 25.5 mM) with or without curcumin (1, 10, or 100 µM). MC3T3-E1 cells were exposed to HG for 24 h and subsequently treated with curcumin (1, 10, or 100 µM) for 48 h. The Levels of ( B ) MDA, ( C ) SOD and ( D ) GSH levels in MC3T3-E1 cells after the above treatment were detected using commercial kits. ( E ) Western blotting analysis of the expression levels of SIRT3, FoxO3a, and antioxidant enzyme in MC3T3-E1 cells. ( F ) Quantitative analysis indicate the expression of the above proteins relative to the control levels, and β-actin is the internal reference. * p < 0.05, *** p < 0.001, **** p < 0.0001 vs. control group, # p < 0.05, ### p < 0.001, #### p < 0.0001 vs. HG group.

Journal: Scientific Reports

Article Title: Curcumin preserves bone health compromised by diabetes by inhibiting osteoporosis through regulation of the SIRT3/FoxO3a signalling pathway

doi: 10.1038/s41598-025-15165-8

Figure Lengend Snippet: Curcumin inhibits high glucose-induced oxidative stress and activates the SIRT3/FoxO3a signaling pathway in MC3T3-E1 cells. ( A ) DCFH-DA staining assay for ROS levels in MC3T3-E1 cells after 48 h of treatment with high glucose (HG, 25.5 mM) with or without curcumin (1, 10, or 100 µM). MC3T3-E1 cells were exposed to HG for 24 h and subsequently treated with curcumin (1, 10, or 100 µM) for 48 h. The Levels of ( B ) MDA, ( C ) SOD and ( D ) GSH levels in MC3T3-E1 cells after the above treatment were detected using commercial kits. ( E ) Western blotting analysis of the expression levels of SIRT3, FoxO3a, and antioxidant enzyme in MC3T3-E1 cells. ( F ) Quantitative analysis indicate the expression of the above proteins relative to the control levels, and β-actin is the internal reference. * p < 0.05, *** p < 0.001, **** p < 0.0001 vs. control group, # p < 0.05, ### p < 0.001, #### p < 0.0001 vs. HG group.

Techniques: Staining, Western Blot, Expressing, Control

Curcumin ameliorates high glucose-induced mitochondrial oxidative stress damage by activating the SIRT3/FoxO3a signaling pathway in MC3T3-E1 cells. ( A ) MC3T3-E1 cells were transfected with SIRT3 shRNA for 24 h, treated with curcumin (100 µM), and subsequently incubated in high glucose (HG, 25.5 mM). Cell viability was assessed using the CCK-8 assay. ( B ) Western blot analysis of SIRT3 protein levels in MC3T3-E1 cells after transfection treatment, with related ( C ) quantitative analysis. ( D ) Western blot analysis of FoxO3a and antioxidant enzyme expression levels in MC3T3-E1 cells, with related ( E ) quantitative analysis. ( F ) JC-1 fluorescence intensity was measured by flow cytometry to evaluate changes in mitochondrial membrane potential. Values are expressed as mean ± standard deviation from three independent experiments ( n = 3). * p < 0.05, ** p < 0.01 vs. control group; # p < 0.05, ### p < 0.001 vs. HG group.

Journal: Scientific Reports

Article Title: Curcumin preserves bone health compromised by diabetes by inhibiting osteoporosis through regulation of the SIRT3/FoxO3a signalling pathway

doi: 10.1038/s41598-025-15165-8

Figure Lengend Snippet: Curcumin ameliorates high glucose-induced mitochondrial oxidative stress damage by activating the SIRT3/FoxO3a signaling pathway in MC3T3-E1 cells. ( A ) MC3T3-E1 cells were transfected with SIRT3 shRNA for 24 h, treated with curcumin (100 µM), and subsequently incubated in high glucose (HG, 25.5 mM). Cell viability was assessed using the CCK-8 assay. ( B ) Western blot analysis of SIRT3 protein levels in MC3T3-E1 cells after transfection treatment, with related ( C ) quantitative analysis. ( D ) Western blot analysis of FoxO3a and antioxidant enzyme expression levels in MC3T3-E1 cells, with related ( E ) quantitative analysis. ( F ) JC-1 fluorescence intensity was measured by flow cytometry to evaluate changes in mitochondrial membrane potential. Values are expressed as mean ± standard deviation from three independent experiments ( n = 3). * p < 0.05, ** p < 0.01 vs. control group; # p < 0.05, ### p < 0.001 vs. HG group.

Techniques: Transfection, shRNA, Incubation, CCK-8 Assay, Western Blot, Expressing, Fluorescence, Flow Cytometry, Membrane, Standard Deviation, Control

Curcumin promotes osteogenic differentiation by activating the SIRT3/FoxO3a signaling pathway in MC3T3-E1 cells. ( A ) MC3T3-E1 cells were transfected with SIRT-3 shRNA for 24 h, treated with curcumin (100 µM), and finally cultured in high glucose (HG, 25.5 mM). Osteoprotegerin (OPG) and osteocalcin (OCN) protein levels in MC3T3-E1 cells were detected by western blotting. ( B ) Quantitative analysis. ( C ) ALP protein ELISA assay. ( D ) ARS staining analysis. ** p < 0.01 vs. control group, # p < 0.05 vs. HG group.

Journal: Scientific Reports

Article Title: Curcumin preserves bone health compromised by diabetes by inhibiting osteoporosis through regulation of the SIRT3/FoxO3a signalling pathway

doi: 10.1038/s41598-025-15165-8

Figure Lengend Snippet: Curcumin promotes osteogenic differentiation by activating the SIRT3/FoxO3a signaling pathway in MC3T3-E1 cells. ( A ) MC3T3-E1 cells were transfected with SIRT-3 shRNA for 24 h, treated with curcumin (100 µM), and finally cultured in high glucose (HG, 25.5 mM). Osteoprotegerin (OPG) and osteocalcin (OCN) protein levels in MC3T3-E1 cells were detected by western blotting. ( B ) Quantitative analysis. ( C ) ALP protein ELISA assay. ( D ) ARS staining analysis. ** p < 0.01 vs. control group, # p < 0.05 vs. HG group.

Techniques: Transfection, shRNA, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Control

Curcumin upregulates the expression of the oxidative stress-related proteins Nrf2 and SIRT3 in bone tissue cells. Histograms indicate the SIRT3 protein expression levels relative to the control group. Values are expressed as mean ± standard deviation for three independent experiments ( n = 3). * p < 0.05, *** p < 0.001 vs. control group, # p < 0.05, ## p < 0.01 vs. HG group.

Journal: Scientific Reports

Article Title: Curcumin preserves bone health compromised by diabetes by inhibiting osteoporosis through regulation of the SIRT3/FoxO3a signalling pathway

doi: 10.1038/s41598-025-15165-8

Figure Lengend Snippet: Curcumin upregulates the expression of the oxidative stress-related proteins Nrf2 and SIRT3 in bone tissue cells. Histograms indicate the SIRT3 protein expression levels relative to the control group. Values are expressed as mean ± standard deviation for three independent experiments ( n = 3). * p < 0.05, *** p < 0.001 vs. control group, # p < 0.05, ## p < 0.01 vs. HG group.

Techniques: Expressing, Control, Standard Deviation

Journal: Scientific Reports

Article Title: Curcumin preserves bone health compromised by diabetes by inhibiting osteoporosis through regulation of the SIRT3/FoxO3a signalling pathway

doi: 10.1038/s41598-025-15165-8

Techniques: Activation Assay

Journal: Scientific Reports

Article Title: Curcumin preserves bone health compromised by diabetes by inhibiting osteoporosis through regulation of the SIRT3/FoxO3a signalling pathway

doi: 10.1038/s41598-025-15165-8

Techniques: Staining, Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: Curcumin preserves bone health compromised by diabetes by inhibiting osteoporosis through regulation of the SIRT3/FoxO3a signalling pathway

doi: 10.1038/s41598-025-15165-8

Techniques: Transfection, shRNA, Incubation, CCK-8 Assay, Western Blot, Expressing, Fluorescence, Flow Cytometry, Membrane, Standard Deviation, Control

Journal: Scientific Reports

Article Title: Curcumin preserves bone health compromised by diabetes by inhibiting osteoporosis through regulation of the SIRT3/FoxO3a signalling pathway

doi: 10.1038/s41598-025-15165-8

Techniques: Transfection, shRNA, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Scientific Reports

Article Title: Curcumin preserves bone health compromised by diabetes by inhibiting osteoporosis through regulation of the SIRT3/FoxO3a signalling pathway

doi: 10.1038/s41598-025-15165-8

Techniques: Expressing, Control, Standard Deviation

A qRT-PCR analysis of transcription factors of GLS2 in EA.hy926 cells treated with Neu5Ac (20 mM) for 12 h. B , C EA.hy926 cells were treated with or without Neu5Ac (20 mM), SIRT3, p-FOXO3a, c-Myc and GLS2 protein expression were assessed by western blot, with quantitative data at right. The molecular docking techniques were utilized to investigate the potential interaction between Neu5Ac and SIRT3, the amino acids that play an important role in the binding of Neu5Ac in the stable interval of molecular dynamics locus were analyzed ( D – F ); the 1000-frame locus of the last 10 ns of molecular dynamics of small molecule Neu5Ac and protein SIRT3 was extracted, and the free binding energy of Neu5Ac and SIRT3 was calculated ( G ). Data were analyzed using unpaired two-tailed Student’s t -tests or one-way ANOVA tests, and presented as the means ± SEM. * p < 0.05 was considered significant, ** p < 0.01, *** p < 0.001.

Journal: Cell Death Discovery

Article Title: N -Acetylneuraminic acid triggers endothelial pyroptosis and promotes atherosclerosis progression via GLS2-mediated glutaminolysis pathway

doi: 10.1038/s41420-024-02233-7

Figure Lengend Snippet: A qRT-PCR analysis of transcription factors of GLS2 in EA.hy926 cells treated with Neu5Ac (20 mM) for 12 h. B , C EA.hy926 cells were treated with or without Neu5Ac (20 mM), SIRT3, p-FOXO3a, c-Myc and GLS2 protein expression were assessed by western blot, with quantitative data at right. The molecular docking techniques were utilized to investigate the potential interaction between Neu5Ac and SIRT3, the amino acids that play an important role in the binding of Neu5Ac in the stable interval of molecular dynamics locus were analyzed ( D – F ); the 1000-frame locus of the last 10 ns of molecular dynamics of small molecule Neu5Ac and protein SIRT3 was extracted, and the free binding energy of Neu5Ac and SIRT3 was calculated ( G ). Data were analyzed using unpaired two-tailed Student’s t -tests or one-way ANOVA tests, and presented as the means ± SEM. * p < 0.05 was considered significant, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies used for immunoblots were as follows: Rabbit Polyclonal Antibody against IL-1β (Beyotime, AF7209, diluted 1:1000), Rabbit Monoclonal Antibody against NLRP3 (Beyotime, AF2155, diluted 1:1000), Rabbit Monoclonal Antibody against Caspase-1 (Beyotime, AF1681, diluted 1:1000), Rabbit Monoclonal Antibody against VCAM-1 (Beyotime, AF1021, diluted 1:1000), Rabbit Polyclonal Antibody against ICAM-1 (Beyotime, AF0195, diluted 1:1000), Rabbit Polyclonal Antibody against GLS2 (ABclonal, A16029, diluted 1:1000), Rabbit Polyclonal Antibody against GSDMD (full length + N-terminal) (ABclonal, A20197, diluted 1:1000), Rabbit Polyclonal Antibody against GSDME (ABclonal, A7432, diluted 1:1000), Rabbit Polyclonal Antibody against IL-18 (ABclonal, A16737, diluted 1:1000), Rabbit Polyclonal Antibody against Phospho-FOXO3A-S253 (ABclonal, AP0684, diluted 1:1000), Rabbit Polyclonal Antibody against c-Myc (Proteintech, 10828-1-AP, diluted 1:1000), mouse Monoclonal Antibody against FOXO3a (Proteintech, 66428-1-Ig, diluted 1:1000), mouse Monoclonal Antibody against SIRT3 (Santa Cruz, sc-365175, diluted 1:1000).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Binding Assay, Two Tailed Test

EA.hy926 cells were preincubated with or without 10058-F4 (60 µM) for 1 h before Neu5Ac (20 mM) treatment for 12 h, GLS2 and DAPI immunostaining were performed, with quantitative data at below; bar = 100 µm ( A , B ); IL-1β, IL-18, Caspase-1, NLRP3, GSDMD-N, GLS2 and ICAM-1 protein expression were assessed by western blot, with quantitative data at below ( C , D ); mitochondrial mass, membrane potential and mitochondrial ROS production were labeled by MitoTracker Green, TMRE and mitoSOX respectively; bar = 31.75 µm ( E ). EA.hy926 cells were transfected with SIRT3 siRNA or NC siRNA before Neu5Ac (20 mM) treatment for 12 h, IL-1β, IL-18, Caspase-1, NLRP3, GSDMD-N, GLS2, p-FOXO3a and ICAM-1 protein expression were assessed by western blot and quantified ( F , G ). Mitochondrial mass, membrane potential and mitochondrial ROS production were labeled by MitoTracker Green, TMRE and mitoSOX respectively; bar = 31.75 µm ( H ). I , J Western blot analysis of IL-1β, IL-18, Caspase-1, NLRP3, GSDMD-N and ICAM-1 of EA.hy926 cells transfected with GLS2 plasmid and SIRT3 siRNA followed by Neu5Ac (20 mM) treatment for 12 h. K EA.hy926 cells were transfected with SIRT3 siRNA or NC siRNA before Neu5Ac (20 mM) treatment for 12 h, Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was performed with an antibody for c-Myc and antibody for IgG as the negative control. Data were analyzed using unpaired two-tailed Student’s t -tests or one-way ANOVA tests, and presented as the means ± SEM. * p < 0.05 was considered significant, ** p < 0.01, *** p < 0.001.

Journal: Cell Death Discovery

Article Title: N -Acetylneuraminic acid triggers endothelial pyroptosis and promotes atherosclerosis progression via GLS2-mediated glutaminolysis pathway

doi: 10.1038/s41420-024-02233-7

Figure Lengend Snippet: EA.hy926 cells were preincubated with or without 10058-F4 (60 µM) for 1 h before Neu5Ac (20 mM) treatment for 12 h, GLS2 and DAPI immunostaining were performed, with quantitative data at below; bar = 100 µm ( A , B ); IL-1β, IL-18, Caspase-1, NLRP3, GSDMD-N, GLS2 and ICAM-1 protein expression were assessed by western blot, with quantitative data at below ( C , D ); mitochondrial mass, membrane potential and mitochondrial ROS production were labeled by MitoTracker Green, TMRE and mitoSOX respectively; bar = 31.75 µm ( E ). EA.hy926 cells were transfected with SIRT3 siRNA or NC siRNA before Neu5Ac (20 mM) treatment for 12 h, IL-1β, IL-18, Caspase-1, NLRP3, GSDMD-N, GLS2, p-FOXO3a and ICAM-1 protein expression were assessed by western blot and quantified ( F , G ). Mitochondrial mass, membrane potential and mitochondrial ROS production were labeled by MitoTracker Green, TMRE and mitoSOX respectively; bar = 31.75 µm ( H ). I , J Western blot analysis of IL-1β, IL-18, Caspase-1, NLRP3, GSDMD-N and ICAM-1 of EA.hy926 cells transfected with GLS2 plasmid and SIRT3 siRNA followed by Neu5Ac (20 mM) treatment for 12 h. K EA.hy926 cells were transfected with SIRT3 siRNA or NC siRNA before Neu5Ac (20 mM) treatment for 12 h, Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was performed with an antibody for c-Myc and antibody for IgG as the negative control. Data were analyzed using unpaired two-tailed Student’s t -tests or one-way ANOVA tests, and presented as the means ± SEM. * p < 0.05 was considered significant, ** p < 0.01, *** p < 0.001.

Techniques: Immunostaining, Expressing, Western Blot, Membrane, Labeling, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Negative Control, Two Tailed Test

Excess endogenous and exogenous Neu5Ac promoted the expression of transcriptional factor c-Myc via SIRT3/FOXO3a signaling pathway, contributing to increased transcription of GLS2 and accelerated glutaminolysis. With increased α-KG production and decreased GSH, Neu5Ac ultimately leads to mitochondrial damage, mitoROS production and pyroptosis in ECs and exacerbates atherosclerosis.

Journal: Cell Death Discovery

Article Title: N -Acetylneuraminic acid triggers endothelial pyroptosis and promotes atherosclerosis progression via GLS2-mediated glutaminolysis pathway

doi: 10.1038/s41420-024-02233-7

Figure Lengend Snippet: Excess endogenous and exogenous Neu5Ac promoted the expression of transcriptional factor c-Myc via SIRT3/FOXO3a signaling pathway, contributing to increased transcription of GLS2 and accelerated glutaminolysis. With increased α-KG production and decreased GSH, Neu5Ac ultimately leads to mitochondrial damage, mitoROS production and pyroptosis in ECs and exacerbates atherosclerosis.

Techniques: Expressing

Figure 2: Conditional deletion of Sirt3 in LysM-Creetargeted cells decreases bone resorption in aged female mice. (AeC) Number of osteoclasts (N.Oc/B.Pm) (A), osteoclast surface (Oc.S/BS) (B) and number of osteoblasts (N.Ob/B.Pm) (C) per endocortical bone surface of nondecalciﬁed femur sections stained for TRAPase activity from 16- month-old female Sirt3DLysM mice and wild-type littermates (n ¼ 17e20 animals/group). (D) qPCR of mRNA isolated from L1 vertebrae (n ¼ 6 animals/group) obtained from 16- month-old female mice. (E) Serum concentration of a collagen degradation product (CTx) and N-terminal propeptide of type I procollagen (P1NP) in 16-month-old female Sirt3DLysM

Journal: Molecular metabolism

Article Title: Mitochondrial protein deacetylation by SIRT3 in osteoclasts promotes bone resorption with aging in female mice.

doi: 10.1016/j.molmet.2024.102012

Figure Lengend Snippet: Figure 2: Conditional deletion of Sirt3 in LysM-Creetargeted cells decreases bone resorption in aged female mice. (AeC) Number of osteoclasts (N.Oc/B.Pm) (A), osteoclast surface (Oc.S/BS) (B) and number of osteoblasts (N.Ob/B.Pm) (C) per endocortical bone surface of nondecalciﬁed femur sections stained for TRAPase activity from 16- month-old female Sirt3DLysM mice and wild-type littermates (n ¼ 17e20 animals/group). (D) qPCR of mRNA isolated from L1 vertebrae (n ¼ 6 animals/group) obtained from 16- month-old female mice. (E) Serum concentration of a collagen degradation product (CTx) and N-terminal propeptide of type I procollagen (P1NP) in 16-month-old female Sirt3DLysM

Article Snippet: Mouse monoclonal antibodies against SIRT3 (1:2000, Santa Cruz Biotechnology, sc-365175) were used to detect SIRT3 proteins.

Techniques: Staining, Activity Assay, Isolation, Concentration Assay

Figure 3: Conditional deletion of Sirt3 in LysM-Creetargeted cells decreases osteoclast mitophagy in aged female mice. BMMs were isolated from 16-month-old female Sirt3DLysM mice and wild-type littermates and were cultured with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for 3 days. (AeB) Mitochondrial and ATP-linked respirations per cell, in osteoclasts, were measured by Seahorse (n ¼ 8e14 wells/group). (C) MitoSOX to evaluate mitochondrial ROS production (n ¼ 6 wells/group). (D) Representative pictures of osteoclasts stained with JC-1 (left), and quantiﬁcation of mitochondrial membrane potential (right) (triplicates of pooled cultures). Scale bar: 500 mm. (E) Ultrastructural appearance of mitochondria of osteoclasts by TEM (left) and the number of cristae formation per mitochondria section (right) (triplicates of pooled cultures). (F) ATP levels (expressed as relative light units, RLU). (GeH) Representative mitochondrial protein levels by Western blot (left) and expression levels as the indicated ratio (right) (triplicates of pooled cultures). (I) Representative PINK1 acetylation levels by Western blot (left) and expression levels as the indicated ratio (right) (triplicate cultures). (J) Tfam levels in mRNA of cultured osteoclasts measured by qPCR (triplicate cultures). Line and error bars represent mean SD. P values were determined using Student’s t-test. All measures were performed in cultured BMMs pooled from 4 to 5 mice/group and repeated at least twice.

Journal: Molecular metabolism

Article Title: Mitochondrial protein deacetylation by SIRT3 in osteoclasts promotes bone resorption with aging in female mice.

doi: 10.1016/j.molmet.2024.102012

Figure Lengend Snippet: Figure 3: Conditional deletion of Sirt3 in LysM-Creetargeted cells decreases osteoclast mitophagy in aged female mice. BMMs were isolated from 16-month-old female Sirt3DLysM mice and wild-type littermates and were cultured with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for 3 days. (AeB) Mitochondrial and ATP-linked respirations per cell, in osteoclasts, were measured by Seahorse (n ¼ 8e14 wells/group). (C) MitoSOX to evaluate mitochondrial ROS production (n ¼ 6 wells/group). (D) Representative pictures of osteoclasts stained with JC-1 (left), and quantiﬁcation of mitochondrial membrane potential (right) (triplicates of pooled cultures). Scale bar: 500 mm. (E) Ultrastructural appearance of mitochondria of osteoclasts by TEM (left) and the number of cristae formation per mitochondria section (right) (triplicates of pooled cultures). (F) ATP levels (expressed as relative light units, RLU). (GeH) Representative mitochondrial protein levels by Western blot (left) and expression levels as the indicated ratio (right) (triplicates of pooled cultures). (I) Representative PINK1 acetylation levels by Western blot (left) and expression levels as the indicated ratio (right) (triplicate cultures). (J) Tfam levels in mRNA of cultured osteoclasts measured by qPCR (triplicate cultures). Line and error bars represent mean SD. P values were determined using Student’s t-test. All measures were performed in cultured BMMs pooled from 4 to 5 mice/group and repeated at least twice.

Article Snippet: Mouse monoclonal antibodies against SIRT3 (1:2000, Santa Cruz Biotechnology, sc-365175) were used to detect SIRT3 proteins.

Techniques: Isolation, Cell Culture, Staining, Membrane, Western Blot, Expressing

Figure 6: Deletion of Sirt3 induces hyperacetylation of mitochondrial proteins in aged osteoclasts. (AeC) BMMs were isolated from 24-month-old female C57BL/6 mice (AeB) or 16-month-old female Sirt3DLysM mice and littermate controls (C) and were cultured with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for 3 days. Representative blots showing the acetylation status of mitochondrial proteins in whole cell lysates by Western blot (left) and expression levels as the indicated ratio (right) (triplicate cultures). (D) Western blot analysis of protein isolated from femoral bone shafts (n ¼ 4 animals/group) obtained from 16-month-old female mice. All Western blot represents a minimum of 3 independent experiments. (EeJ) BMMs were isolated from 16-month-old female Sirt3 null mice and littermate controls and were cultured with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for 3 days. (EeG) Quantitative analysis of the global proteome was performed with label-free tandem mass spectrometry (triplicate cultures). The fold change in total proteins is presented in volcano plots for the effects of RANKL treatment or genotype (E). Top 5 highly up- or down-regulated pathways (RANKL vs Vehicle) (FeG). (HeJ) A comprehensive acetylome analysis was performed with 3 samples per group as described in the ﬂow chart (H). The fold change in hyperacetylated proteins is presented in volcano plots for the effects of RANKL treatment or genotype (I). Top 10 hyper-acetylated proteins (Sirt3 KO vs WT). Asterisk indicates published sites (J). Line and error bars represent mean SD. P values were determined using Student’s t-test.

Journal: Molecular metabolism

Article Title: Mitochondrial protein deacetylation by SIRT3 in osteoclasts promotes bone resorption with aging in female mice.

doi: 10.1016/j.molmet.2024.102012

Figure Lengend Snippet: Figure 6: Deletion of Sirt3 induces hyperacetylation of mitochondrial proteins in aged osteoclasts. (AeC) BMMs were isolated from 24-month-old female C57BL/6 mice (AeB) or 16-month-old female Sirt3DLysM mice and littermate controls (C) and were cultured with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for 3 days. Representative blots showing the acetylation status of mitochondrial proteins in whole cell lysates by Western blot (left) and expression levels as the indicated ratio (right) (triplicate cultures). (D) Western blot analysis of protein isolated from femoral bone shafts (n ¼ 4 animals/group) obtained from 16-month-old female mice. All Western blot represents a minimum of 3 independent experiments. (EeJ) BMMs were isolated from 16-month-old female Sirt3 null mice and littermate controls and were cultured with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for 3 days. (EeG) Quantitative analysis of the global proteome was performed with label-free tandem mass spectrometry (triplicate cultures). The fold change in total proteins is presented in volcano plots for the effects of RANKL treatment or genotype (E). Top 5 highly up- or down-regulated pathways (RANKL vs Vehicle) (FeG). (HeJ) A comprehensive acetylome analysis was performed with 3 samples per group as described in the ﬂow chart (H). The fold change in hyperacetylated proteins is presented in volcano plots for the effects of RANKL treatment or genotype (I). Top 10 hyper-acetylated proteins (Sirt3 KO vs WT). Asterisk indicates published sites (J). Line and error bars represent mean SD. P values were determined using Student’s t-test.

Article Snippet: Mouse monoclonal antibodies against SIRT3 (1:2000, Santa Cruz Biotechnology, sc-365175) were used to detect SIRT3 proteins.

Techniques: Isolation, Cell Culture, Western Blot, Expressing, Mass Spectrometry

$Figure 7: SIRT3-induced ATPIF1 deacetylation activates osteoclast mitophagy in aged mice. (AeE, not B) BMMs from 24-month-old female C57BL/6 mice transduced with lentiviral vectors expressing control sh-RNA or sh-RNAs targeting the hyperacetylated proteins and cultured with RANKL for 5 days (A and C) or 3 days (DeG). (A) Representative pictures (left) and number (right) of TRAPþ multinucleated osteoclasts and Von Kossaestained bone biomaterial surface. (triplicate cultures). Scale bar: 500 mm. (B) BMMs were isolated from 24-month-old female C57BL/6 mice and were cultured with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for 3 days. Western blot analysis of expression of ATPIF1. (triplicate cultures). (C) Levels of mRNA of an osteoclast marker in cultured osteoclasts measured by qPCR. (triplicate cultures). (DeF) Different fractions of mitochondrial respiration per cell measured with Seahorse (n ¼ 10e14 wells/group). (G) Western blot analysis of expression of mitophagy marker. (triplicate cultures). Line and error bars represent mean SD. P values were determined using Student’s t-test. All in vitro assays were performed in cultured BMMs pooled from more than 3 mice and repeated at least twice.$

Journal: Molecular metabolism

Article Title: Mitochondrial protein deacetylation by SIRT3 in osteoclasts promotes bone resorption with aging in female mice.

doi: 10.1016/j.molmet.2024.102012

Figure Lengend Snippet: Figure 7: SIRT3-induced ATPIF1 deacetylation activates osteoclast mitophagy in aged mice. (AeE, not B) BMMs from 24-month-old female C57BL/6 mice transduced with lentiviral vectors expressing control sh-RNA or sh-RNAs targeting the hyperacetylated proteins and cultured with RANKL for 5 days (A and C) or 3 days (DeG). (A) Representative pictures (left) and number (right) of TRAPþ multinucleated osteoclasts and Von Kossaestained bone biomaterial surface. (triplicate cultures). Scale bar: 500 mm. (B) BMMs were isolated from 24-month-old female C57BL/6 mice and were cultured with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for 3 days. Western blot analysis of expression of ATPIF1. (triplicate cultures). (C) Levels of mRNA of an osteoclast marker in cultured osteoclasts measured by qPCR. (triplicate cultures). (DeF) Different fractions of mitochondrial respiration per cell measured with Seahorse (n ¼ 10e14 wells/group). (G) Western blot analysis of expression of mitophagy marker. (triplicate cultures). Line and error bars represent mean SD. P values were determined using Student’s t-test. All in vitro assays were performed in cultured BMMs pooled from more than 3 mice and repeated at least twice.

Article Snippet: Mouse monoclonal antibodies against SIRT3 (1:2000, Santa Cruz Biotechnology, sc-365175) were used to detect SIRT3 proteins.

Techniques: Transduction, Expressing, Control, Cell Culture, Isolation, Western Blot, Marker, In Vitro

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