Journal: Cell death & disease

Article Title: Oroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization.

doi: 10.1038/cddis.2015.86

Figure Lengend Snippet: Figure 1 OA inhibits glycolysis by upregulating SIRT3 and SIRT3-mediated destabilization of HIF1α. (a) MDA-MB-231 cells were treated with OA under conditions of hypoxia or normoxia for 10 h. Glucose uptake was measured using the Amplex Red assay, and production of lactic acid was assayed with the Lactic Acid production Detection kit. (b) SIRT3-deficient MDA-MB-231 cells were incubated with 100 μM OA for 10 h under hypoxia. Glucose uptake and production of lactic acid were assayed as above. (c, d) The protein expressions (c) and mRNA expressions (d) of SIRT3, HIF1α and HK II was detected by immunoblotting and quantitative RT-PCR, respectively, in cells treated with OA for 10 h under hypoxia. (e, f) SIRT3-deficient cells were treated with/without OA for 10 h under hypoxia. The protein expressions of SIRT3, HIF1α and HK II (e) and the mRNA expression of HK II (f) were detected. (g–i) HIF1α overexpressed MDA-MB-231 cells were then incubated with 100 μM OA for 10 h under conditions of hypoxia. Glucose uptake and production of lactic acid (g), the protein expression (h) and the mRNA expression (i) of HK II were assayed. Bars, S.D.; *Po0.05 or **Po0.01 versus untreated controls in hypoxia

Article Snippet: The HIF1α human cDNA clone and SIRT3 human cDNA clone were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Amplex Red Assay, Incubation, Western Blot, Quantitative RT-PCR, Expressing

Journal: Cell death & disease

Article Title: Oroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization.

doi: 10.1038/cddis.2015.86

Figure Lengend Snippet: Figure 2 OA destabilized HIF1α by influencing SIRT3-modulated activity of PDH. (a) MDA-MB-231 cells were treated with NAM (10 mM) for 10 h under hypoxic conditions. HIF1α was immunoprecipitated using SIRT3 or SIRT1 antibodies. Western blot assays were performed for HIF1α, SIRT1 and SIRT3. (b) For 10 h OA treatment under hypoxia, the expression of PHD was detected by immunoblotting. (c) MDA-MB-231 cells were treated with OA for 10 h under hypoxic or normoxia conditions. The expression of hydroxylated HIF1α (HIF-OH) was detected by immunoblotting. (d) MDA-MB-231 cells treated with or without 100 μM OA or 3 μM MG-132 or 1 mM DMOG for 10 h as indicated were immunoblotted against antibodies specific to HIF-OH, or total HIF1α. (e) SIRT3-deficient MDA-MB-231 cells were incubated with 100 μM OA for 10 h under hypoxia. HIF-OH was detected by immunoblotting. (f) SIRT3-deficient MDA-MB-231 cells were incubated with or without 100 μM OA or 1 mM DMOG for 10 h under hypoxia. Hydroxylated HIF1α (HIF-OH) and total HIF1α were detected by immunoblotting

Article Snippet: The HIF1α human cDNA clone and SIRT3 human cDNA clone were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Expressing, Incubation

Journal: Cell death & disease

Article Title: Oroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization.

doi: 10.1038/cddis.2015.86

Figure Lengend Snippet: Figure 3 OA-induced activation of PDHs through the regulation of ·O2 −levels by SIRT3. (a) MDA-MB-231 cells were treated with OA for 10 h under hypoxia. The levels of cellular superoxide anion (·O2 −) and hydrogen peroxide (H2O2) were detected by FACSCalibur flow cytometry using the fluorescent dye dihydroethidium at Ex/Em of 300/610 nm or dichlorofluorescein-diacetate at Ex/Em of 488/525 nm, respectively. (b) SIRT3-deficient MDA-MB-231 cells were incubated with or without 100 μM OA for 10 h under hypoxia, and ·O2 −was detected. (c, d) Cells were treated with 100 μM OA or 10 mM N-acetyl cysteine (antioxidant used as a positive control) in the presence of 1 μM rotenone for 10 h under hypoxia. The levels of ·O2 −(c), protein expression of HIF-OH and HIF1α (d) were detected. (e, f) Cells were treated with 100 μM OA with/without 1 μM rotenone for 10 h under hypoxia. Cell survival rate (e), glucose uptake and production of lactic acid (f) were detected, respectively. Bars, S.D.; *Po0.05 or **Po0.01 versus untreated control

Article Snippet: The HIF1α human cDNA clone and SIRT3 human cDNA clone were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Activation Assay, Flow Cytometry, Incubation, Positive Control, Expressing, Control

Journal: Cell death & disease

Article Title: Oroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization.

doi: 10.1038/cddis.2015.86

Figure Lengend Snippet: Figure 4 The regulations of OA on glycolysis and HIF1α were associated with increased SOD2 levels under hypoxia. (a–c) MDA-MB-231 cells were treated with OA for 10 h under hypoxia. The protein expression (a), mRNA expression (b) and activity (c) of SOD2 were detected by immunoblotting, Cu/Zn-SOD and Mn-SOD Assay Kit, and quantitative RT-PCR, respectively. (d, e) SIRT3-deficient MDA-MB-231 cells were incubated with or without 100 μM OA for 10 h under hypoxia conditions. The protein expression (d) and activity (e) of SOD2 were detected as described above. (f, g) SOD2-deficient MDA-MB-231 cells were incubated with or without 100 μM OA for 10 h under hypoxia. Glucose uptake and production of lactic acid (f), and protein expressions of HIF-OH, HIF1α, and HKII (g) were detected. (h) MDA-MB-231 cells were treated with OA for 10 h under hypoxia. SOD2 was immunoprecipitated using SIRT3 antibody, and acetylated-SOD2 (Ac-SOD2) was immunoprecipitated using SOD2 antibody in mitochondrial extracts. (i–k) MDA-MB-231 cells were treated with or without 100 μM OA or 2 μM TAS or 10 mM NAM for 10 h under hypoxia. (i) Ac-SOD2 was immunoprecipitated using SOD2 antibody in mitochondrial extracts. (j) The activity of SOD2 was detected. (k) The protein expressions of SOD2, HIF-OH, HIF1α, and HK II was detected by immunoblotting. (l, m) SIRT3- deficient MDA-MB-231 cells were incubated with or without 100 μM OA or 10 mM NAM for 10 h under hypoxia. Ac-SOD2 was immunoprecipitated in mitochondrial extracts (l), and the activity of SOD2 (m) was assayed. Bars, S.D.; *Po0.05 or **Po0.01 versus untreated controls

Article Snippet: The HIF1α human cDNA clone and SIRT3 human cDNA clone were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Expressing, Activity Assay, Western Blot, Quantitative RT-PCR, Incubation, Immunoprecipitation

Journal: Cell death & disease

Article Title: Oroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization.

doi: 10.1038/cddis.2015.86

Figure Lengend Snippet: Figure 5 OA regulated the mRNA expression of SOD2 through the interaction of SIRT3 with FOXO3a in mitochondria. (a–d) MDA-MB-231 cells were treated with OA for 10 h under hypoxia conditions. (a) Mitochondria were isolated after treatment and subjected to western blot analysis for FOXO3a. (b) FOXO3a was immunoprecipitated using a SIRT3 antibody. (c) ChIP analysis of FOXO3a binding to the SOD2 promoter in MDA-MB-231 cells treated with OA. Cells were fixed with 1% formaldehyde to crosslink protein–DNA interactions, sonicated, and fixed cells were immunoprecipitated with anti-FOXO3a antibody. DNA was eluted and purified before analysis using specific primers and quantitative RT- PCR. (d) Nuclei were isolated after treatment and subjected to EMSA to assess the binding of FOXO3a to the SOD2 promoter in mitochondria. (e) SIRT3-deficient and normal MDA- MB-231 cells were treated with OA or 10 mM NAM for 10 h under hypoxia conditions. SOD2 promoter luciferase reporter plasmid (p3x-FOXO3a-luc) was transfected into MDA-MB-231 cells. Luciferase activity was normalized to Renilla activity and expressed as luciferase/Renilla relative units. Bars, S.D.; *Po0.05 or **Po0.01 versus untreated controls

Article Snippet: The HIF1α human cDNA clone and SIRT3 human cDNA clone were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Expressing, Isolation, Western Blot, Immunoprecipitation, Binding Assay, Sonication, Purification, Quantitative RT-PCR, Luciferase, Plasmid Preparation, Transfection, Activity Assay

Journal: The Journal of Cell Biology

Article Title: Drosophila Sirt2/mammalian SIRT3 deacetylates ATP synthase β and regulates complex V activity

doi: 10.1083/jcb.201404118

Figure Lengend Snippet: Human ATP synthase β is an acetylated protein, and its deacetylation is regulated by SIRT3. (A) pCMV vector or ATP synthase β (DDK tagged) was transfected in HEK293T cells, immunoprecipitated using an antibody to DDK tag, and probed with an antibody to acetyl-Lys (Ac-Lys). (B) HEK293T cells were cotransfected with ATP synthase β (ATP syn β) and either SIRT3 siRNA or scrambled siRNA. ATP synthase β was immunoprecipitated, and its acetylation status was assessed. The bottom blot shows reduction of SIRT3 protein upon siRNA treatment. Knockdown of SIRT3 increases acetylation of ATP synthase β. (C) Expression vector for wild-type SIRT3 was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. Overexpression of SIRT3 decreases acetylation of ATP synthase β. (D) HEK293T cells were cotransfected with ATP synthase β and either SIRT4 siRNA or scrambled siRNA. SIRT4 knockdown does not affect acetylation of ATP synthase β. (E) Wild-type SIRT4 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT4 overexpression does not affect acetylation of ATP synthase β. (F) HEK293T cells were cotransfected with ATP synthase β and either SIRT5 siRNA or scrambled siRNA. SIRT5 knockdown does not affect acetylation of ATP synthase β. (G) Wild-type SIRT5 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT5 overexpression does not affect acetylation of ATP synthase β. (H) HEK293T cells were cotransfected with ATP synthase β and either SIRT1 siRNA or scrambled siRNA. SIRT1 knockdown does not affect acetylation of ATP synthase β. (I) Wild-type SIRT1 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT1 overexpression does not affect acetylation of ATP synthase β. (J) Mitochondria were prepared from SIRT3 siRNA–treated or scrambled siRNA–treated cells, and complex V activity was measured. The activity of mitochondria from scrambled siRNA treatment was taken as 100%. SIRT3 knockdown results in an ∼40% decrease in complex V activity. n = 3; error bars represent SDs. **, P ≤ 0.01–0.001 in Student’s t test. (K) Endogenous ATP synthase β was immunoprecipitated from HEK293T cells overexpressing SIRT3, and the immunoprecipitate was probed with antibodies to ATP synthase β and SIRT3. SIRT3 can coimmunoprecipitate with ATP synthase β. IP, immunoprecipitation; WB, Western blot.

Article Snippet: DDK-tagged (similar to FLAG tag) ATP synthase β (RC201638) and DDK-tagged human SIRT3 (RC200190), SIRT4 (RC212226), SIRT5 (RC200189), and SIRT1 (RC218134) plasmids were obtained from OriGene.

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Knockdown, Expressing, Over Expression, Activity Assay, Western Blot

Journal: The Journal of Cell Biology

Article Title: Drosophila Sirt2/mammalian SIRT3 deacetylates ATP synthase β and regulates complex V activity

doi: 10.1083/jcb.201404118

Figure Lengend Snippet: Acetylation of ATP synthase β at Lys 259 and Lys 480 regulates complex V activity. (A) Nondegradable (non-deg) ATP synthase β (ATP syn β) is resistant to targeted siRNA-mediated degradation. (B) siRNA-resistant versions of ATP synthase β wherein Lys 259 or Lys 480 either individually or together were substituted with Arg or Gln and cotransfected with siRNA to ATP synthase β. Mitochondria were prepared, and complex V activity was measured using an immunocapture assay followed by the amount of ATP synthase β in the same samples. The activity of siRNA-resistant ATP synthase β is taken as 100%. Substitution of either Lys or both with Arg results in increased activity, whereas substitution with Gln results in decreased complex V activity. **, P ≤ 0.01–0.001; ***, P ≤ 0.001–0.0001. (C) An overview of the crystal structure of bovine mitochondrial F1–stator complex is shown on the left in ribbon representation. The F1 domain contains 3α (green), 3β (purple), and a single subunit of γ (pink). The stator complex shows portions of subunit b (teal), oligomycin sensitivity-conferring protein (orange), and F6 (yellowish green). The right shows a closer view of the region around the active site (marked by the black box in the left image). The Lys residues are shown as spheres, and the active site amino acids are shown as stick models. Acetylation of Lys 259 (Lys 206 in the crystal structure) and Lys 480 (430 in the crystal structure) could affect protein conformation near the active site. (D) Endogenous ATP synthase β was immunoprecipitated from human breast cancer cell lines, and its acetylation status was assessed using an acetyl-Lys antibody. ATP synthase β is more acetylated in MDA-MB-231 cells compared with T47D. (E) Complex V activity was measured in mitochondria prepared from human breast cancer cell lines. The activity is significantly less in MDA-MB-231 cells compared with that in T47D cells. n = 3. Analysis of variance was performed, and Tukey’s honest significance test was applied to determine significance. T47D–MDA-MB-231: adjusted P = 1.0 × 10 −7 ; T47D–MDA-MB-435: adjusted P = 1.9 × 10 −5 . (F) Oxygen consumption is less in MDA-MB-231 compared with that in T47D mitochondria. n = 3. Analysis of variance was performed, and Tukey’s honest significance test was applied to determine significance. T47D–MDA-MB-231: adjusted P = 2.0 × 10 −6 ; T47D–MDA-MB-435: adjusted P = 1.0 × 10 −5 . (G) A model depicting Drosophila Sirt2/mammalian SIRT3-mediated deacetylation of ATP synthase β and its impact on complex V activity. Error bars represent SDs. IP, immunoprecipitation; WB, Western blot.

Article Snippet: DDK-tagged (similar to FLAG tag) ATP synthase β (RC201638) and DDK-tagged human SIRT3 (RC200190), SIRT4 (RC212226), SIRT5 (RC200189), and SIRT1 (RC218134) plasmids were obtained from OriGene.

Techniques: Activity Assay, Immunoprecipitation, Western Blot

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA ( siSIRT3 ) and subjected to immunoblotting analysis. b Quantification of protein levels in ( a ). n = 8–10 independent experiments. c HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA ( siSIRT3 ) and subjected to Q-PCR analysis to detect mRNA levels of GATA2 and EDN1 . n = 8 independent experiments. d The interactions between SIRT3 and FUNDC1 or GATA2 in HUVECs were determined by immunoprecipitation (IP) and immunoblotting (IB) analysis. e The co-localization of SIRT3 and FUNDC1 or GATA2 were determined by immunofluorescence staining. Nuclei were stained by DAPI. MERGE1 indicates merge of SIRT3 and DAPI; MERGE2 indicates merge of FUNDC1, SIRT3, and DAPI; MERGE3 indicates merge of GATA2 and DAPI; MERGE4 indicates merge of GATA2, SIRT3, and DAPI. Scale bar: 10 µm. f HUVECs were transfected with control siRNA (siCtrl) or FUNDC1 siRNA ( siFUNDC1 ) in the presence of BSA or 200 µM PA and subjected to immunofluorescence staining analysis. MERGE1 indicates merge of SIRT3 and DAPI; MERGE2 indicates merge of FUNDC1, SIRT3, and DAPI. Scale bar: 10 µm. g Pearson’s coefficient calculated by ZEN software, indicates the co-localization of FUNDC1 and SIRT3. n = 9 cells from three independent experiments. h Quantification of nuclear SIRT3 level. i HUVECs were transfected with control siRNA (siCtrl) or FUNDC1 siRNA ( siFUN ) in the presence of BSA or 200 µM PA. Non-nuclear fraction (Non-Nuc), nuclear fraction (Nuc), and mitochondria fraction (Mito) were prepared and subjected to immunoblotting analysis. n = 9 cells from three independent experiments. j Quantification of protein level of SIRT3 long isoform (SIRT3-L) in the nucleus (Nuclear SIRT3-L) and in mitochondria (Mitochondrial SIRT3-L). n = 4 independent experiments. k HUVECs were double transfected with control siRNA (siCtrl) or FUNDC1 ( siFUN ) and SIRT3 siRNA and subjected to immunoblotting analysis. l Quantification of GATA2 protein level in ( k ). n = 5 independent experiments. m HUVECs were double transfected with either control siRNA (siCtrl) or FUNDC1 and SIRT3 siRNA and subjected to Q-PCR analysis to detect END1 mRNA level. n = 8 independent experiments. Data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05.

Article Snippet: 500 μg of protein was incubated with antibodies against SIRT3 (Cell Signaling Technology, 5490), GATA2 (Novus, NBP1-82581), or acetylated-Lysine (Cell Signaling Technology, 9441) at 4 °C overnight.

Techniques: Transfection, Control, Western Blot, Immunoprecipitation, Immunofluorescence, Staining, Software

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA and then treated with 50 µg/ml cycloheximide (CHX) for 240 min. Cell lysates were subjected to immunoblotting analysis. b Quantification of GATA2 protein level in ( a ). n = 4 independent experiments. c Acetylated GATA2 level was determined by IP of acetylate lysine (Ac-lysine) in cells transfected with SIRT3 siRNA and followed by immunoblotting (IB) of GATA2. d Quantification of the enrichment of acetylated GATA2 in ( c ). n = 4 independent experiments. e Acetylated GATA2 level was determined by IP of Ac-lysine in cells treated with PA (200 µM) and followed by immunoblotting (IB) GATA2. f Quantification of the enrichment of acetylated GATA2 in ( e ). n = 4 independent experiments. g HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA ( siSIRT3 ) and subjected to immunoblotting analysis. h HUVECs were transfected with control Myc-Flag-tagged plasmid ( Myc-Flag-Ctrl ) or Myc-Flag-SIRT3 plasmid ( Myc-Flag-SIRT3 ) and subjected to immunoblotting analysis. i , j Quantification of protein levels of FUNDC1. n = 6–9 independent experiments. k HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA ( siSIRT3 and subjected to Q-PCR analysis to detect FUNDC1 mRNA level. n = 8. l HUVECs were transfected with control siRNA (siCtrl) or SIRT3 siRNA and then treated with 50 µg/ml cycloheximide (CHX) for 240 min. Cell lysates were subjected to immunoblotting analysis. m Quantification of GATA2 protein level in ( l ). n = 3 independent experiments. Data are presented as mean ± SD, in ( b and m ), star represents the comparison between groups siCtrl and siSIRT3 at the same treatment intervals, pound represents the comparison with control group (time = 0 min). * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. p > 0.05. # p < 0.05, ## p < 0.01, ### p < 0.001, n.s. p > 0.05.

Article Snippet: 500 μg of protein was incubated with antibodies against SIRT3 (Cell Signaling Technology, 5490), GATA2 (Novus, NBP1-82581), or acetylated-Lysine (Cell Signaling Technology, 9441) at 4 °C overnight.

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, Comparison

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: a Potential KFERQ-like motifs of human FUNDC1 and SIRT3. b Interactions between HSC70 with FUNDC1 and SIRT3-L in HUVECs were detected by IP and IB. c Co-staining of FUNDC1 with HSC70 and SIRT3-L by IF. d Interaction of SIRT3-L and FUNDC1 was analyzed in HUVECs transfected with HSC70 siRNA. e Quantification of protein levels in HUVECS transfected with HSC70 siRNA. f Quantification of the binding activity of FUNDC1 with SIRT3-L in HSC70 knockdown cells, indicated by the enrichment of FUNDC1. n = 4 independent experiments. g HUVECs were transfected with control siRNA (siCtrl) or HSC70 siRNA ( siHSC70 ) in the presence of BSA or 200 µM PA. Non-nuclear fraction (Non-Nuc), nuclear fraction (Nuc), and mitochondria fraction (Mito) were prepared and subjected to immunoblotting analysis. h Quantification of protein level of SIRT3 long isoform (SIRT3-L) in the nucleus (Nuclear SIRT3-L) and in mitochondria (Mitochondrial SIRT3-L). n = 4 independent experiments. Data are presented as mean ± SD, * p < 0.05, ** p < 0.01 derived from Student’s t tests.

Article Snippet: 500 μg of protein was incubated with antibodies against SIRT3 (Cell Signaling Technology, 5490), GATA2 (Novus, NBP1-82581), or acetylated-Lysine (Cell Signaling Technology, 9441) at 4 °C overnight.

Techniques: Staining, Transfection, Binding Assay, Activity Assay, Knockdown, Control, Western Blot, Derivative Assay

Journal: Nature Communications

Article Title: Endothelial FUNDC1 regulates metabolic reprogramming and the obesity-diabetes transition through the SIRT3/GATA2/endothelin-1 axis

doi: 10.1038/s41467-026-68548-4

Figure Lengend Snippet: In the healthy state, endothelial FUNDC1 anchors SIRT3-L in mitochondria, limiting its nuclear accumulation and regulating its interaction with GATA2, thereby maintaining normal ET-1 production and preserving vascular and metabolic homeostasis. Under overnutrition-induced metabolic stress, SIRT3-L is released from GATA2, contributing to enhanced GATA2-mediated ET-1 transcription. Elevated ET-1 promote angiogenesis to provide the space for adipocyte hyperplasia at the early stage and later exacerbate insulin resistance and thus driving the progression from obesity to T2DM. Importantly, SIRT3-L translocation to mitochondria also facilitates FUNDC1 degradation at the early obese stage, which triggers a compensatory increase in FUNDC1 transcription and ultimately results in FUNDC1 overexpression in a late-stage. The elevation of FUNDC1 further enhances mitochondrial recruitment of SIRT3-L, amplifying ET-1 production. In contrast, in Fundc1 EC-specific knockout (ECKO) mice, the absence of FUNDC1 prevents mitochondrial recruitment of SIRT3-L, leading to its nuclear retention, where SIRT3-L promotes GATA2 degradation and suppresses ET-1 transcription. The loss of Fundc1 in ECs not only suppresses angiogenesis at early overnutrition stage but also prevents EC senescence at late stage to attenuate the potential diabetic vascular complications.

Article Snippet: 500 μg of protein was incubated with antibodies against SIRT3 (Cell Signaling Technology, 5490), GATA2 (Novus, NBP1-82581), or acetylated-Lysine (Cell Signaling Technology, 9441) at 4 °C overnight.

Techniques: Preserving, Translocation Assay, Over Expression, Knock-Out

Journal: iScience

Article Title: Increased SIRT3 combined with PARP inhibition rescues motor function of SBMA mice.

doi: 10.1016/j.isci.2023.107375

Figure Lengend Snippet: Figure 1. SIRT3 and SOD2 proteins are reduced in AR100Q mice, and activating and increasing SIRT3 reduces ROS and cell death in vitro (A) Western analysis of quadriceps from 11 week-old WT, AR24Q, and AR100Q male mice (n = 3). Top, total SOD2 with tubulin loading control, and densitometry analysis of total SOD2 compared to tubulin. Bottom, a second SDS-PAGE gel probed for acetylated (acetyl-K68) SOD2 with tubulin loading control, and densitometry analysis of acetyl-K68/total SOD2 (from A top) after each were normalized to tubulin loading control. (B) Western analysis of quadriceps from 11 week-old WT, AR24Q, and AR100Q male mice (n = 3). Endogenous SIRT3 and tubulin loading control with densitometry analysis of SIRT3 normalized to tubulin loading control. (C) ROS levels in AR112Q expressing PC12 cells (n = 12) treated with 10 nM DHT plus either DMSO (vehicle control), 1 mM viniferin, 1 mM honokiol, or 100 mM melatonin for 48 h (representative image from 3 experiments).

Article Snippet: Primary antibodies used include: mouse a-tubulin IgG1 (Sigma-Aldrich, T6199), mouse-myc IgG1 (Thermo Fisher, R950-25), mouse SOD2-A2 IgG2b (Santa Cruz, sc-133134), rabbit acetyl K68 SOD2 IgG (Abcam, ab137037), mouse anti-Poly-ADP-Ribose IgG3 (Trevigen, 4335-MC-100), rabbit SIRT3 (Cell Signaling, D22A3), rabbit mAb androgen receptor (Cell Signaling, D6F11), mouse ANTI-FLAG M2 (Sigma, F1804).

Techniques: In Vitro, Western Blot, Control, SDS Page, Expressing

Journal: iScience

Article Title: Increased SIRT3 combined with PARP inhibition rescues motor function of SBMA mice.

doi: 10.1016/j.isci.2023.107375

Figure Lengend Snippet: Figure 2. Overexpression of flag-tagged SIRT3 (mouse isoform 1) in AR100Q mice increases quadriceps muscle mass but does not rescue motor function. Exogenous SIRT3 and total SOD2 proteins are reduced in the AR100Q quadriceps (A) Muscle mass (left) and percent of total body mass (right) of quadriceps from WT, SIRT3, AR100Q, and AR100Q-SIRT3 male mice (n = 3) at age 8 weeks. (B) Behavioral cohort composed of 10 X WT, 10 X SIRT3 only, 10 X AR100Q, and 8 X AR100Q-SIRT3 male mice underwent analysis as indicated. Top, accelerating rotarod analysis was performed weekly, starting at age 6 weeks. Bottom, accelerating treadmill analysis at ages 8, 11, and 12 weeks. * = difference between WT vs. AR100Q and AR100Q-SIRT3. (C) Left, immunoblots of endogenous SIRT3 and flag-tagged SIRT3, and tubulin loading control in quadriceps muscle of WT, SIRT3, AR100Q, and AR100Q- SIRT3 male mice (n = 3) at age 11 weeks. Note that endogenous SIRT3 cannot be seen in WT or AR100Q because of early exposure time. Right, densitometry analysis of flag-tagged SIRT3 normalized to tubulin loading control. * = Two-way t-test, p % 0.05. (D) Left, immunoblots of total and acetylated (acetyl-K68) SOD2 and tubulin loading controls (2 separate gels) in quadriceps muscle of WT, SIRT3, AR100Q, and AR100Q-SIRT3 male mice (n = 3) at age 11 weeks. Right, densitometry analysis of total SOD2/tubulin and acetyl-K68/total SOD2 after each were normalized to tubulin loading control. (E) Acetylome analysis of SOD2 acetyl-K68 (left) and acetyl-K122 (right) intensity of quadriceps from WT, SIRT3, AR24Q, AR100Q, and AR100Q-SIRT3 male mice (n = 3) at age 11 weeks. (n = 2 for SIRT3 and AR100Q-SIRT3 due to outliers below the 3 correlation Z score cut off).

Article Snippet: Primary antibodies used include: mouse a-tubulin IgG1 (Sigma-Aldrich, T6199), mouse-myc IgG1 (Thermo Fisher, R950-25), mouse SOD2-A2 IgG2b (Santa Cruz, sc-133134), rabbit acetyl K68 SOD2 IgG (Abcam, ab137037), mouse anti-Poly-ADP-Ribose IgG3 (Trevigen, 4335-MC-100), rabbit SIRT3 (Cell Signaling, D22A3), rabbit mAb androgen receptor (Cell Signaling, D6F11), mouse ANTI-FLAG M2 (Sigma, F1804).

Techniques: Over Expression, Western Blot, Control

Journal: iScience

Article Title: Increased SIRT3 combined with PARP inhibition rescues motor function of SBMA mice.

doi: 10.1016/j.isci.2023.107375

Figure Lengend Snippet: Figure 3. Targeting diminished NAD+ with PARP inhibition in cells and skeletal muscle harboring polyQ-expanded AR (A) Levels of NAD+ in quadriceps (left) and gastrocnemius (right) from WT, AR24Q, and AR100Q male mice (n = 3) at age 11 weeks. (B) Levels of NAD+ in quadriceps (left) and gastrocnemius (right) from WT, SIRT3, AR100Q, and AR100Q-SIRT3 male mice (n = 3) at age 11 weeks. (C) Levels of NAD+ in AR10Q-, and AR112Q-expressing PC12 cells (n = 3) treated with EtOH DHT, or DHT + olaparib (representative image from 3 experiments). (D) ROS levels of PC12 polyQ-expanded AR112Q expressing cells (n = 12) treated with either DMSO (vehicle control), or 1 mM olaparib for 48 h (representative image from 3 experiments). *** = Nested two-way t-test, p % 0.001. (E) Cell death of AR100Q expressing C2C12 myotubes (n = 3) treated with either DMSO (vehicle control), or 1 mM olaparib for 10 days (representative image from 3 experiments). * = Nested two-way t-test, p % 0.05. (F) Muscle mass (left) and percent of total mass (right) of gastrocnemius from WT and AR100Q male mice (n = 3) at age 8 weeks fed no-drug compounded or olaparib-compounded food. (G) Levels of NAD+ in quadriceps (left) and gastrocnemius (right) from WT and AR100Q male mice (n = 4) at age 8 weeks fed no-drug compounded food or olaparib-compounded food. (A–G) All statistical analysis show mean G SD, and used a two-way ANOVA and Tukey’s post-hoc (* = p % 0.05, ** = p % 0.01, *** = p % 0.001) unless otherwise noted. EtOH, ethanol; WT, wild type; SIRT3, SIRT3-flag overexpression; AR100Q, polyQ-expanded AR; AR100Q-SIRT3, polyQ-expanded AR with SIRT3-flag overexpression; AR10Q, non-polyQ-expanded AR; AR112Q, polyQ-expanded AR; olaparib, PARP inhibitor (1150 g of olaparib in 1 kg of feed).

Article Snippet: Primary antibodies used include: mouse a-tubulin IgG1 (Sigma-Aldrich, T6199), mouse-myc IgG1 (Thermo Fisher, R950-25), mouse SOD2-A2 IgG2b (Santa Cruz, sc-133134), rabbit acetyl K68 SOD2 IgG (Abcam, ab137037), mouse anti-Poly-ADP-Ribose IgG3 (Trevigen, 4335-MC-100), rabbit SIRT3 (Cell Signaling, D22A3), rabbit mAb androgen receptor (Cell Signaling, D6F11), mouse ANTI-FLAG M2 (Sigma, F1804).

Techniques: Inhibition, Expressing, Control, Over Expression

Journal: iScience

Article Title: Increased SIRT3 combined with PARP inhibition rescues motor function of SBMA mice.

doi: 10.1016/j.isci.2023.107375

Figure Lengend Snippet: Figure 4. Overexpressing SIRT3 plus PARP inhibition with olaparib further reduces ROS in vitro and rescues treadmill running without affecting AR100Q protein levels (A) ROS in AR112Q SIRT3-MYC-overexpressing PC12 cells (n = 12) treated with either DMSO (vehicle control), or 1 mM olaparib for 48 h with (right) or without (left) ROS induction with 250 mM TBHP (representative image from 3 experiments). (B) Muscle mass and percent of total mass of quadriceps (left) and gastrocnemius (right) from WT, AR100Q, and AR100Q-SIRT3 male mice (n = 3) at age 8 weeks fed no-drug compounded or olaparib-compounded feed. (C) Behavioral cohort composed of 8 X WT fed no-drug compounded food, 11 X AR100Q fed no-drug compounded food, and 11 X AR100Q-SIRT3 male mice fed olaparib-compounded food. Left, accelerating rotarod analysis starting at age 6 weeks; * = difference between WT vs. AR100Q and AR100Q-SIRT3. Right, accelerating treadmill analysis at 11 weeks of age. (D) Left, immunoblot of total PAR following a PAR affinity resin assay from 8-week-old mouse quadriceps. WT lanes are oversaturated. Right-top, analysis of total protein lysates utilized in the PAR assay showing AR100Q monomer, and SDS-insoluble HMW aggregated AR100Q and tubulin loading control. Right- bottom, densitometry analysis of AR monomer and SDS-insoluble HMW aggregated AR normalized to tubulin loading control. (A–D) All statistical analysis show mean G SD, and used a two-way ANOVA and Tukey’s post-hoc (* = p % 0.05, ** = p % 0.01, *** = p % 0.001). TBHP, tert- butyl hydroperoxide; WT, wild type; AR100Q, polyQ-expanded AR; AR100Q-SIRT3, polyQ-expanded AR with SIRT3-flag overexpression; HMW, high molecular weight; olaparib, PARP inhibitor (1150 g of olaparib in 1 kg of feed); PAR, poly-ADP-ribose.

Article Snippet: Primary antibodies used include: mouse a-tubulin IgG1 (Sigma-Aldrich, T6199), mouse-myc IgG1 (Thermo Fisher, R950-25), mouse SOD2-A2 IgG2b (Santa Cruz, sc-133134), rabbit acetyl K68 SOD2 IgG (Abcam, ab137037), mouse anti-Poly-ADP-Ribose IgG3 (Trevigen, 4335-MC-100), rabbit SIRT3 (Cell Signaling, D22A3), rabbit mAb androgen receptor (Cell Signaling, D6F11), mouse ANTI-FLAG M2 (Sigma, F1804).

Techniques: Inhibition, In Vitro, Control, Western Blot, Over Expression, High Molecular Weight

Journal: iScience

Article Title: Increased SIRT3 combined with PARP inhibition rescues motor function of SBMA mice.

doi: 10.1016/j.isci.2023.107375

Figure Lengend Snippet: Figure 5. Acetylome analysis reveals substantially increased acetylation of peptides within AR100Q mouse quadriceps, with highly dysregulated metabolic pathways that are partially corrected by overexpression of SIRT3 WT, SIRT3-M1-flag (SIRT3), AR24Q, AR100Q, AR100Q-SIRT3 male mice (n = 3) were aged to 11 weeks. Quadriceps from these mice were digested with trypsin and acetylated peptides were enriched using Cell Signaling PTMScan Acetyl-Lysine Motif antibody. Enriched peptides were analyzed by LC-MS/MS on a Q Ex active HF mass spectrometer. For analysis n = 2 for SIRT3 and AR100Q-SIRT3 due to outliers below the 3 correlation Z score cut off. (A) The levels of total acetylated sites in peptides are indicated by the intensity values. Statistical analysis shows mean G SD; *** = two-way ANOVA and Tukey’s post-hoc, p % 0.001. (B) Differential expression of acetylated proteins between groups. Limma was used to find protein acetylation significantly different between groups. Direction of fold changes are listed in the top row. The number of proteins within the false discovery rate of <5% (estimated % of genes that satisfy f-value threshold that may be false positives) are listed in the bottom row. (C) Heatmap of the top 30 proteins significantly changed in AR100Q and AR100Q-SIRT3 quadriceps compared to WT, SIRT3Q, and AR24Q controls. Left, upregulated acetylation of proteins. Right, downregulated acetylation of proteins. Red = increased acetylation. Blue = decreased acetylation.

Article Snippet: Primary antibodies used include: mouse a-tubulin IgG1 (Sigma-Aldrich, T6199), mouse-myc IgG1 (Thermo Fisher, R950-25), mouse SOD2-A2 IgG2b (Santa Cruz, sc-133134), rabbit acetyl K68 SOD2 IgG (Abcam, ab137037), mouse anti-Poly-ADP-Ribose IgG3 (Trevigen, 4335-MC-100), rabbit SIRT3 (Cell Signaling, D22A3), rabbit mAb androgen receptor (Cell Signaling, D6F11), mouse ANTI-FLAG M2 (Sigma, F1804).

Techniques: Over Expression, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Quantitative Proteomics

Journal: iScience

Article Title: Increased SIRT3 combined with PARP inhibition rescues motor function of SBMA mice.

doi: 10.1016/j.isci.2023.107375

Figure Lengend Snippet: Figure 6. Overexpressing SIRT3 plus PARP inhibition with olaparib fully restores hexokinase activity in quadriceps of AR100Q mice Levels of Hexokinase in quadriceps from WT, AR100Q, and AR100Q-SIRT3 male mice (n = 3) at age 11 weeks fed no-drug compounded or olaparib-compounded food. All statistical analysis show mean G SD, and used a two-way ANOVA and Tukey’s post-hoc (*** = p % 0.001). WT, wild type; SIRT3, SIRT3-flag overexpression; AR100Q, polyQ-expanded AR; AR100Q-SIRT3, polyQ-expanded AR with SIRT3-flag overexpression; olaparib, PARP inhibitor (1150 g of olaparib in 1 kg of feed).

Article Snippet: Primary antibodies used include: mouse a-tubulin IgG1 (Sigma-Aldrich, T6199), mouse-myc IgG1 (Thermo Fisher, R950-25), mouse SOD2-A2 IgG2b (Santa Cruz, sc-133134), rabbit acetyl K68 SOD2 IgG (Abcam, ab137037), mouse anti-Poly-ADP-Ribose IgG3 (Trevigen, 4335-MC-100), rabbit SIRT3 (Cell Signaling, D22A3), rabbit mAb androgen receptor (Cell Signaling, D6F11), mouse ANTI-FLAG M2 (Sigma, F1804).

Techniques: Inhibition, Activity Assay, Over Expression

Journal: Aging (Albany NY)

Article Title: α-Mangostin remodels visceral adipose tissue inflammation to ameliorate age-related metabolic disorders in mice

doi: 10.18632/aging.102512

Figure Lengend Snippet: α-Man blocks MAPKs and NF-κB pathways and activates SIRT3 in eWAT from LPS-treated mice. ( A ) The expression of iNOS and SIRT3 in eWAT were detected by Western blot analyses. ( B ) The expression of p-ERK, ERK, p-p38 and p38 were detected by Western blot. ( C ) The expression of p-IKKα/β, IKKα, IKKβ, p-IκBα, IκBα, p-p65 and p65 were detected by Western blot. α-Tubulin was used as an internal control. Data are expressed as means ± SD ( n = 5). # P < 0.05 LPS vs. control, * P < 0.05, LPS + α-Man vs. LPS.

Article Snippet: The shRNA targeting SIRT3 (mouse, sc-61556-SH), control shRNA plasmid-A (mouse, sc-108060), and shRNA transfection reagent (mouse, sc-108061) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Control

Journal: Aging (Albany NY)

Article Title: α-Mangostin remodels visceral adipose tissue inflammation to ameliorate age-related metabolic disorders in mice

doi: 10.18632/aging.102512

Figure Lengend Snippet: Effects of α-Man in LPS stimulated SIRT3-knockdown RAW264.7 macrophages. ( A ) The protein expression of SIRT3 was determined by Western blot in LPS-induced RAW264.7 macrophages. α-Tubulin was used as an internal loading control. Data are normalized to the mean value of LPS group. ( B ) NO production was determined by Griess reagent. ( C ) iNOS abundance was measured by Western blot. α-Tubulin was used as an internal loading control. Data are normalized to the mean value of scrambled LPS group. The levels of IL-6 ( D ), TNF-α ( E ) and MCP-1 ( F ) were determined by ELISA kit. Data are shown as means ± SD ( n = 5). ## P < 0.01, LPS vs. DMSO, ** P < 0.01, α-Man + LPS vs. LPS, & P < 0.05, SIRT3KD LPS vs. scrambled LPS, $ P < 0.05, $$ P < 0.01, SIRT3KD α-Man vs. scrambled α-Man.

Article Snippet: The shRNA targeting SIRT3 (mouse, sc-61556-SH), control shRNA plasmid-A (mouse, sc-108060), and shRNA transfection reagent (mouse, sc-108061) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Knockdown, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Aging (Albany NY)

Article Title: α-Mangostin remodels visceral adipose tissue inflammation to ameliorate age-related metabolic disorders in mice

doi: 10.18632/aging.102512

Figure Lengend Snippet: α-Man mitigates age-related adipose tissue inflammation through NF-κB and MAPKs pathways. ( A ) Relative mRNA levels of iNos , Il-1β and Tnf-α in eWAT were analyzed by qRT-PCR. ( B ) The protein levels of iNOS, COX-2 and SIRT3 in eWAT were detected by Western blot analyses and quantified using Image J. ( C ) The expression of p-IKKα/β, IKKα, IKKβ, p-IκBα, IκBα, p-p65 and p65 was detected by Western blot. ( D ) The expression of p-ERK, ERK, p-p38, p38, p-JNK and JNK were detected by Western blot. α-Tubulin was used as an internal control. Data are normalized to the mean value of old group. Data are expressed as means ± SD ( n = 5). # P < 0.05, ## P < 0.01, old mice vs. young mice, * P < 0.05, ** P < 0.01, α-Man vs. old mice. Y, young mice; O, old mice; L, old mice administrated with 25 mg/kg α-Man; H, old mice administrated with 50 mg/kg α-Man.

Article Snippet: The shRNA targeting SIRT3 (mouse, sc-61556-SH), control shRNA plasmid-A (mouse, sc-108060), and shRNA transfection reagent (mouse, sc-108061) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Control

Journal: Antioxidants (Basel, Switzerland)

Article Title: Nicotinamide Riboside and Phycocyanin Oligopeptides Affect Stress Susceptibility in Chronic Corticosterone-Exposed Rats.

doi: 10.3390/antiox12101849

Figure Lengend Snippet: Figure 5. Effects of NR and PC oligopeptides on liver SIRT1 (A), SIRT3 (B), and NAMPT (C) levels in rats subject to chronic corticosterone (CORT). The densitometric analysis of the relative intensity according to the control group of the Western blotting bands was performed with β-actin normal- ization to ensure equal protein loading (D). Blots were repeated at least three times (n = 3), and a representative blot is shown. Data are expressed as a percent of the control set at 100%. The error bars above the lines indicate the standard deviation of the mean. Different symbols (a–e) indicate significant differences among the groups (ANOVA and Tukey post hoc test; p < 0.05). SIRT1, sir- tuin 1; SIRT3, sirtuin 3; NAMPT, nicotinamide phosphoribosyltransferase. CS: chronic stress; NR: Nicotinamide riboside (26.44 mg/kg); PC-LD: Phycocyanin oligopeptide, low dose (2.64 mg/kg); PC-HD: Phycocyanin oligopeptide, high dose (26.44 mg/kg). Rats except those of the control group were given daily corticosterone injections (40 mg/kg) to induce stress conditions, or NR and PC were orally administered for 21 days. Full immunoblots presented in Figure S3.

Article Snippet: NC membranes were blocked with 5% bovine serum albumin for 2 h. The membranes were incubated with rat-specific primary antibodies diluted to 1:1000 (IL-6 (sc-57315), TNF-α (sc-52746), IL-1β (sc-515598), TRF2 (sc47693), Tin2 (sc-73177), SIRT3 (sc-365175), NAMPT (sc-393444) (Santa Cruz Biotechnology, Heidelberg, Germany), IL-8 (NBP2-16958), POTlb (NBP2-50255) (Novus Biotech, CO, USA), POT1a (PA5-75366), SIRT1 (MA5-27217), and TRF1 (MA1-46375) (Thermo Fisher Scientific, Waltham, MA, USA)) overnight at 4 ◦C.

Techniques: Control, Western Blot, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Atractylodin inhibits ferroptosis in sepsis-induced acute gastrointestinal injury via SIRT3/PRDX3

doi: 10.3892/mmr.2025.13695

Figure Lengend Snippet: Atractylodin suppresses the expression of Ac-PRDX3 while inducing the expression of SIRT3 in stomach and colon tissues. (A) The western blot assay of Ac-PRDX3 and PRDX3 in stomach tissue and (E) colon tissue of each group. Quantifying relative protein expression of Ac-PRDX3/PRDX3 in the (B) stomach and (F) colon tissue. The western blot assay of SIRT3 in (C) stomach and (G) colon tissue of each group. Quantification of relative protein expression of SIRT3/GAPDH in the (D) stomach and (H) colon tissue. Confocal immunofluorescence of the mice cochlear tissues in different groups. Confocal immunofluorescence of SIRT3 (green) in (I) stomach tissue and (K) colon tissue; scale bar, 50 µm. the cell nuclear parts were labeled with blue. Quantification of relative expression of SIRT3 in the (J) stomach and (L) colon tissue. Co-IP of PRDX3 with SIRT3 in (M) stomach and (N) colon tissue. (O-R) Relative quantitative evaluation of the western-blot analysis for SIRT3/PRDX3, PRDX3/GAPDH expression was obtained using ImageJ software. Values are means ± SD of n=3 (A-R) experiments. *P<0.05, **P<0.01 vs. the model group, ## P<0.01 vs. the Sham group. PRDX3, peroxiredoxin-3; SIRT3, NAD-dependent protein deacetylase sirtuin-3, mitochondrial; Co-IP, coimmunoprecipitation.

Article Snippet: The membrane was blocked with 5% BSA in 1X PBST buffer (0.01% Tween-20) for 90 min at room temperature and was probed with GAPDH) antibody (1:5,000; Proteintech Group, Inc., 10494-1-AP) or GPX4 antibody (1:1,000; Abcam, ab125066), Mitochondrial import receptor subunit TOM20 homolog (1:1,000; Abcam, ab186735), transferrin receptor protein 1 (TFR1) antibody (1:1,000; Abcam, ab214039), PRDX3 antibody (1:1,000; Abcam, ab73349), SIRT3 antibody (1:1,000; Proteintech Group, Inc., 10099-1-AP), SLC7A11 antibody (1:1,000; Proteintech Group, Inc., 32384-1-AP), occludin antibody (1:1,000; Abcam, ab216327), and zona occludens protein 1 (ZO-1) antibody (1:1,000; Proteintech Group, Inc.; cat. no. 21773-1-AP), for 2 h at room temperature.

Techniques: Expressing, Western Blot, Immunofluorescence, Labeling, Co-Immunoprecipitation Assay, Software, Histone Deacetylase Assay

Journal: Molecular Medicine Reports

Article Title: Atractylodin inhibits ferroptosis in sepsis-induced acute gastrointestinal injury via SIRT3/PRDX3

doi: 10.3892/mmr.2025.13695

Figure Lengend Snippet: Regulatory mechanism of SIRT3 and PRDX3 in sepsis-induced ferroptosis-mediated mitochondrial oxidative damage. (A) Western blot analysis of SIRT3 protein level in the stomach and colon tissue. Mice were injected with sh-SIRT3 or control shRNA (shRNA-NC). (B) Western blot analysis of GPX4 protein level in stomach and (C) Western blot analysis of GPX4 protein level in colon tissue. (D) Relative SIRT3/GAPDH expression obtained using ImageJ software. Relative quantitative evaluation of the GPX4/GAPDH expression in (E) stomach and (F) colon tissue of each group. (G) Western blot analysis of TOM20, occludin, and ZO-1 protein level in stomach tissue of each group. Relative quantitative evaluation of (H) TOM20, (I) occludin and (J) ZO-1 protein expression level in stomach tissue in each group. (K) Western blot analysis of TOM20, occludin, and ZO-1 protein level in stomach and colon tissue of each group. Relative quantitative evaluation of (L) TOM20, (M) occludin and (N) ZO-1 protein expression level in colon tissue. Activity of mitochondrial respiratory chain complex (O) I, mitochondrial respiratory chain complex II (P), mitochondrial respiratory chain complex III (Q), mitochondrial respiratory chain complex IV (R), mitochondrial respiratory chain complex V (S) in the stomach tissue. (T-X) The activity of mitochondrial respiratory chain complex I (T), mitochondrial respiratory chain complex II (U), mitochondrial respiratory chain complex III (V), mitochondrial respiratory chain complex IV (W), mitochondrial respiratory chain complex V (X) in the colon tissue. Values are means ± SD of n=3 (A-N) or n=6 (O-X) experiments. **P<0.01. SIRT3, NAD-dependent protein deacetylase sirtuin-3, mitochondrial; PRDX3, peroxiredoxin-3; sh, short hairpin; GPX4, Phospholipid hydroperoxide glutathione peroxidase; TOM20, mitochondrial import receptor subunit TOM20 homolog; ZO-1, zona occludens protein 1.

Article Snippet: The membrane was blocked with 5% BSA in 1X PBST buffer (0.01% Tween-20) for 90 min at room temperature and was probed with GAPDH) antibody (1:5,000; Proteintech Group, Inc., 10494-1-AP) or GPX4 antibody (1:1,000; Abcam, ab125066), Mitochondrial import receptor subunit TOM20 homolog (1:1,000; Abcam, ab186735), transferrin receptor protein 1 (TFR1) antibody (1:1,000; Abcam, ab214039), PRDX3 antibody (1:1,000; Abcam, ab73349), SIRT3 antibody (1:1,000; Proteintech Group, Inc., 10099-1-AP), SLC7A11 antibody (1:1,000; Proteintech Group, Inc., 32384-1-AP), occludin antibody (1:1,000; Abcam, ab216327), and zona occludens protein 1 (ZO-1) antibody (1:1,000; Proteintech Group, Inc.; cat. no. 21773-1-AP), for 2 h at room temperature.

Techniques: Western Blot, Injection, Control, shRNA, Expressing, Software, Activity Assay, Histone Deacetylase Assay

Journal: Genetics Research

Article Title: Sirt3 Regulates Response to Oxidative Stress by Interacting with BER Proteins in Colorectal Cancer

doi: 10.1155/2022/7299555

Figure Lengend Snippet: Acetylated lysine level in tested proteins after incubation with a mixture of Sirt3 and NAD ( ∗∗∗ — p < 0.01; ∗ — p < 0.05).

Article Snippet: Silencing Sirt3 gene: cells were treated with Sirt3 shRNA Lentiviral Particles Transduction (Santa Cruz) according to manufacturer's instruction.

Techniques: Incubation

Journal: Genetics Research

Article Title: Sirt3 Regulates Response to Oxidative Stress by Interacting with BER Proteins in Colorectal Cancer

doi: 10.1155/2022/7299555

Figure Lengend Snippet: Coimmunoprecipitation of Sirt3 with MUTYH, NEIL1, and APE1 proteins and lack of coimmunoprecipitation with NEIL2 and LIG3. Coimmunoprecipitation section shows the presence or absence of physical interaction after incubation with antibodies against the indicated protein (with IgG as negative control) followed by incubation with anti-Sirt3. Input section shows the presence of protein after treatment with indicated antibody without coimmunoprecipitation.

Article Snippet: Silencing Sirt3 gene: cells were treated with Sirt3 shRNA Lentiviral Particles Transduction (Santa Cruz) according to manufacturer's instruction.

Techniques: Incubation, Negative Control

Journal: Genetics Research

Article Title: Sirt3 Regulates Response to Oxidative Stress by Interacting with BER Proteins in Colorectal Cancer

doi: 10.1155/2022/7299555

Figure Lengend Snippet: Effect of Sirt3 gene silencing and overexpression on the levels of Sirt3, APE1, NEIL1, and MUTYH proteins in conditions of oxidative stress induced by antimycin A (at final concentrations of 10 μ M and 100 μ M). The control for the Sirt3 silenced and overexpression groups are indicative of the protein level with respect to the wild type. The percentage level is the change in the protein level after a given amount of time with respect to the control. The statistically significant difference ( p < 0.05) between the increase/decrease in protein level with respect to the wild-type is shown in bold.

Article Snippet: Silencing Sirt3 gene: cells were treated with Sirt3 shRNA Lentiviral Particles Transduction (Santa Cruz) according to manufacturer's instruction.

Techniques: Over Expression, Control, Incubation

Journal: Genetics Research

Article Title: Sirt3 Regulates Response to Oxidative Stress by Interacting with BER Proteins in Colorectal Cancer

doi: 10.1155/2022/7299555

Figure Lengend Snippet: The effect of silencing/overexpressing the Sirt3 gene on the level of BER proteins in total and mitochondrial protein after induction of oxidative stress with antimycin A (at final concentrations of 10 μ M and 100 μ M). The control for the Sirt3 silenced and overexpression groups are indicative of the protein level with respect to the wild type. The percentage level is the change in the protein level after a given amount of time with respect to the control. ∗ The statistically significant difference ( p < 0.05) between the increase/decrease in protein level with respect to the wild-type.

Article Snippet: Silencing Sirt3 gene: cells were treated with Sirt3 shRNA Lentiviral Particles Transduction (Santa Cruz) according to manufacturer's instruction.

Techniques: Control, Over Expression

Journal: Genetics Research

Article Title: Sirt3 Regulates Response to Oxidative Stress by Interacting with BER Proteins in Colorectal Cancer

doi: 10.1155/2022/7299555

Figure Lengend Snippet: The level of apoptosis in cells exposed to the damaging factor (ionizing radiation at a dose of 25 Gy) in case of silencing/overexpressing of the Sirt3 gene ( ∗ — p < 0.05).

Article Snippet: Silencing Sirt3 gene: cells were treated with Sirt3 shRNA Lentiviral Particles Transduction (Santa Cruz) according to manufacturer's instruction.

Techniques: