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OriGene full length murine traf6 cdna sequence
Full Length Murine Traf6 Cdna Sequence, supplied by OriGene, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/traf6+cdna/Traf6+(NM_009424)+Mouse+Tagged+ORF+Clone/10__1016_slash_j__bonr__2026__101923-75-15-22
Average 94 stars, based on 4 article reviews

full length murine traf6 cdna sequence - by Bioz Stars, 2026-09

94/100 stars

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Plasmid Preparation:

Article Title: Down-regulation of miR-146b-5p by long noncoding RNA MALAT1 in hepatocellular carcinoma promotes cancer growth and metastasis.
Article Snippet: The human immortal liver cell line LO2 and four HCC cell lines (MHCC97-H, SMMC-7721, Hep3B and HepG2) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and cultured with appropriate conditions. miR-146b-5p expression vector (HmiR0164MR04), miRNA control vector (CmiR0001-MR04, miR-NC), miR-146b-5p inhibitor (HmiR-AN0197AM01, anti-miR-146b), miRNA control inhibitor (CmiR-AN0001-AM01, anti-miR-NC) were purchased from GeneCopoeia Co. Ltd. (Guangzhou, China). .. TRAF6 cDNA clone vector (SC109844) was provided by OriGene Co. Ltd. (Beijing, China). .. TRAF6 siRNA (s14389), MALAT1 siRNA (4455877) and negative control siRNA (AM4611) were purchased from Applied Biosystems (Foster City, CA, USA).

Article Title: Down-regulation of miR-146b-5p by long noncoding RNA MALAT1 in hepatocellular carcinoma promotes cancer growth and metastasis
Article Snippet: The human immortal liver cell line LO2 and four HCC cell lines (MHCC97-H, SMMC-7721, Hep3B and HepG2) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and cultured with appropriate conditions. miR-146b-5p expression vector (HmiR0164-MR04), miRNA control vector (CmiR0001-MR04, miR-NC), miR-146b-5p inhibitor (HmiR-AN0197-AM01, anti-miR-146b), miRNA control inhibitor (CmiR-AN0001-AM01, anti-miR-NC) were purchased from GeneCopoeia Co. Ltd. (Guangzhou, China). .. TRAF6 cDNA clone vector (SC109844) was provided by OriGene Co. Ltd. (Beijing, China). .. TRAF6 siRNA (s14389), MALAT1 siRNA (4455877) and negative control siRNA (AM4611) were purchased from Applied Biosystems (Foster City, CA, USA).

Image Search Results

(A) Luciferase activity, normalized to GFP intensity, of Min6 β-cells transfected with empty vector (EV), NFκB, RELB, IRF3, STAT3, STAT1, Type I Interferon, Type II Interferon, IFNB1, MAPK, TFEB, C/EBPβ, ATF4, and p53 reporter plasmids following exposure to vehicle (DMSO + BSA), LPS (5 nM for 2 h), or palmitate (0.5 mM for 48 h). n = 3/group. ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 2-way ANOVA. (B) qPCR analysis of Traf6 , normalized to Hprt expression, (left) and TRAF6 protein expression by western blot (WB), quantified by densitometry (right), in CRISPR- generated sgScram or sgTraf6 Min6 β-cells. Cyclophilin B (PPIB) served as a loading control. n = 4/group. **** P < 0.0001 by Student’s unpaired t-test. ( C) Luciferase activity, normalized to GFP intensity, of sgScram and sgTraf6 Min6 cells transfected with reporter plasmids following exposure to vehicle or 5 nM LPS for 2 h. n = 3/group. *** P < 0.001, **** P < 0.0001 by 2-way ANOVA. (D) Luciferase activity, normalized to GFP intensity, of sgScram and sgTraf6 Min6 cells transfected with reporter plasmids following exposure to vehicle or 0.5 mM palmitate for 48 h. n = 3/group. **** P < 0.0001 by 2-way ANOVA.

Journal: bioRxiv

Article Title: TRAF6 integrates innate immune signals to regulate glucose homeostasis via Parkin-dependent and -independent mitophagy

doi: 10.1101/2025.01.31.635900

Figure Lengend Snippet: (A) Luciferase activity, normalized to GFP intensity, of Min6 β-cells transfected with empty vector (EV), NFκB, RELB, IRF3, STAT3, STAT1, Type I Interferon, Type II Interferon, IFNB1, MAPK, TFEB, C/EBPβ, ATF4, and p53 reporter plasmids following exposure to vehicle (DMSO + BSA), LPS (5 nM for 2 h), or palmitate (0.5 mM for 48 h). n = 3/group. ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 2-way ANOVA. (B) qPCR analysis of Traf6 , normalized to Hprt expression, (left) and TRAF6 protein expression by western blot (WB), quantified by densitometry (right), in CRISPR- generated sgScram or sgTraf6 Min6 β-cells. Cyclophilin B (PPIB) served as a loading control. n = 4/group. **** P < 0.0001 by Student’s unpaired t-test. ( C) Luciferase activity, normalized to GFP intensity, of sgScram and sgTraf6 Min6 cells transfected with reporter plasmids following exposure to vehicle or 5 nM LPS for 2 h. n = 3/group. *** P < 0.001, **** P < 0.0001 by 2-way ANOVA. (D) Luciferase activity, normalized to GFP intensity, of sgScram and sgTraf6 Min6 cells transfected with reporter plasmids following exposure to vehicle or 0.5 mM palmitate for 48 h. n = 3/group. **** P < 0.0001 by 2-way ANOVA.

Article Snippet: Constructs for co-immunoprecipitation overexpression studies included pCMV5-FLAG-TRAF6 , pCMV3-C-HA-ECSIT (Sino Biological, Catalog#HG14497-CY), pRK5-Myc-Parkin , and pcDNA3.1 3x-HA-NRDP1 .

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, CRISPR, Generated, Control

(A) A schematic diagram illustrating Cre-mediated recombination at the Traf6 locus. (B) Protein expression and densitometry quantification (relative to Control RFD) of TRAF6 by WB in islets isolated from control or Traf6 Δβ mice fed regular fat diet (RFD) or high fat diet (HFD) for 15 weeks. PPIB served as a loading control. n = 4/group. * P < 0.05, **** P < 0.0001 by 1-way ANOVA. (C) Total body mass of male control or Traf6 Δβ mice following 30 weeks of RFD or HFD. n = 7-10/group. (D) Blood glucose concentrations measured during intraperitoneal glucose tolerance test (IPGTT; top) and area under the curve (AUC; bottom) of male control and Traf6 Δβ mice fed either RFD (dashed lines) or HFD (solid lines) for 15 weeks (left) or 30 weeks (right). n = 8-12/group. ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 2-way ANOVA (IPGTT) or 1-way ANOVA (AUC). (E) Serum insulin levels measured during in vivo glucose-stimulated insulin release assays in male control and Traf6 Δβ mice fed RFD or HFD for 15 weeks (left) or 30 weeks (right). n = 8-10/group. * P < 0.05, **** P < 0.0001 by 1-way ANOVA. (F) Glucose-stimulated insulin secretion from islets isolated from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks (left) or 30 weeks (right). n = 6-8/group. ** P < 0.01, **** P < 0.0001 by 2-way ANOVA. (G) KCl-stimulated insulin secretion from islets isolated from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. n = 4/group. (H) Insulin content in islets from control or Traf6 Δβ mice fed 15-week RFD or HFD. n = 5-8/group. (I) Pancreatic β-cell mass from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks (left) or 30 weeks (right). n = 5/group. (J) Glucose-stimulated insulin secretion following static incubations in 2 mM and 16.7 mM glucose, performed in human islets treated with DMSO or 1 μM C25-140 (TRAF6 inhibitor) for 24 h and exposed to glucolipotoxicity (GLT; 25 mM glucose + 0.5 mM palmitate) or control (5 mM glucose + BSA) for 48 h. n = 4-5/group. * P < 0.05, **** P < 0.0001 by 2-way ANOVA. (K) Insulin content in human islets treated with DMSO or 1 μM C25-140 for 24 h and exposed to GLT or control for 48 h. n = 4-5/group.

Journal: bioRxiv

Article Title: TRAF6 integrates innate immune signals to regulate glucose homeostasis via Parkin-dependent and -independent mitophagy

doi: 10.1101/2025.01.31.635900

Figure Lengend Snippet: (A) A schematic diagram illustrating Cre-mediated recombination at the Traf6 locus. (B) Protein expression and densitometry quantification (relative to Control RFD) of TRAF6 by WB in islets isolated from control or Traf6 Δβ mice fed regular fat diet (RFD) or high fat diet (HFD) for 15 weeks. PPIB served as a loading control. n = 4/group. * P < 0.05, **** P < 0.0001 by 1-way ANOVA. (C) Total body mass of male control or Traf6 Δβ mice following 30 weeks of RFD or HFD. n = 7-10/group. (D) Blood glucose concentrations measured during intraperitoneal glucose tolerance test (IPGTT; top) and area under the curve (AUC; bottom) of male control and Traf6 Δβ mice fed either RFD (dashed lines) or HFD (solid lines) for 15 weeks (left) or 30 weeks (right). n = 8-12/group. ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 2-way ANOVA (IPGTT) or 1-way ANOVA (AUC). (E) Serum insulin levels measured during in vivo glucose-stimulated insulin release assays in male control and Traf6 Δβ mice fed RFD or HFD for 15 weeks (left) or 30 weeks (right). n = 8-10/group. * P < 0.05, **** P < 0.0001 by 1-way ANOVA. (F) Glucose-stimulated insulin secretion from islets isolated from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks (left) or 30 weeks (right). n = 6-8/group. ** P < 0.01, **** P < 0.0001 by 2-way ANOVA. (G) KCl-stimulated insulin secretion from islets isolated from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. n = 4/group. (H) Insulin content in islets from control or Traf6 Δβ mice fed 15-week RFD or HFD. n = 5-8/group. (I) Pancreatic β-cell mass from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks (left) or 30 weeks (right). n = 5/group. (J) Glucose-stimulated insulin secretion following static incubations in 2 mM and 16.7 mM glucose, performed in human islets treated with DMSO or 1 μM C25-140 (TRAF6 inhibitor) for 24 h and exposed to glucolipotoxicity (GLT; 25 mM glucose + 0.5 mM palmitate) or control (5 mM glucose + BSA) for 48 h. n = 4-5/group. * P < 0.05, **** P < 0.0001 by 2-way ANOVA. (K) Insulin content in human islets treated with DMSO or 1 μM C25-140 for 24 h and exposed to GLT or control for 48 h. n = 4-5/group.

Techniques: Expressing, Control, Isolation, In Vivo

$(A) Oxygen consumption rate (OCR) fractional change (left) and steady state quantification (right) following exposure to 3 mM glucose, 20 mM glucose, 10 μM oligomycin, and 3 mM KCN in isolated islets from control or Traf6 Δβ mice fed 15-week RFD or HFD via Barofuse respirometry. n = 4-6/group. **** P < 0.0001 by 2-way ANOVA. (B) ATP/ADP ratio measured in isolated islets from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. n = 4/group. *** P < 0.001, **** P < 0.0001 by 2-way ANOVA. (C) mtDNA to nuclear DNA ratio in isolated islets from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks by qPCR. n = 5/group. * P < 0.05 by 1-way ANOVA. (D) Citrate synthase activity in isolated islets from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. n = 3/group. * P < 0.05 by 1-way ANOVA. (E) WB for OXPHOS subunits, TOM20, and MFN2 with densitometry (relative to Control RFD) in islets from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. Vinculin (VCL) or PPIB served as loading controls. n = 4/group. * P < 0.05, **** P < 0.0001 by 2-way ANOVA. (F) Representative transmission electron microscopy (TEM) image of β-cells from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks with quantification of mitochondrial morphology (∼100 independent mitochondria scored/animal). n = 3/group. * P < 0.05, ** P < 0.01, *** P < 0.001 by 1-way ANOVA. Scale bars: 500 nM.$

Journal: bioRxiv

Article Title: TRAF6 integrates innate immune signals to regulate glucose homeostasis via Parkin-dependent and -independent mitophagy

doi: 10.1101/2025.01.31.635900

Figure Lengend Snippet: (A) Oxygen consumption rate (OCR) fractional change (left) and steady state quantification (right) following exposure to 3 mM glucose, 20 mM glucose, 10 μM oligomycin, and 3 mM KCN in isolated islets from control or Traf6 Δβ mice fed 15-week RFD or HFD via Barofuse respirometry. n = 4-6/group. **** P < 0.0001 by 2-way ANOVA. (B) ATP/ADP ratio measured in isolated islets from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. n = 4/group. *** P < 0.001, **** P < 0.0001 by 2-way ANOVA. (C) mtDNA to nuclear DNA ratio in isolated islets from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks by qPCR. n = 5/group. * P < 0.05 by 1-way ANOVA. (D) Citrate synthase activity in isolated islets from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. n = 3/group. * P < 0.05 by 1-way ANOVA. (E) WB for OXPHOS subunits, TOM20, and MFN2 with densitometry (relative to Control RFD) in islets from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. Vinculin (VCL) or PPIB served as loading controls. n = 4/group. * P < 0.05, **** P < 0.0001 by 2-way ANOVA. (F) Representative transmission electron microscopy (TEM) image of β-cells from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks with quantification of mitochondrial morphology (∼100 independent mitochondria scored/animal). n = 3/group. * P < 0.05, ** P < 0.01, *** P < 0.001 by 1-way ANOVA. Scale bars: 500 nM.

Techniques: Isolation, Control, Activity Assay, Transmission Assay, Electron Microscopy

(A) Differential RNA expression heatmap of selected mitochondrial biogenesis genes in isolated islets from control or Traf6 Δβ mice fed HFD for 15 weeks. n = 3-4/group. (B) Flow cytometry quantification of mitophagy flux by MtPhagy dye approach in β-cells from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. n = 5-6/group. *** P < 0.001 by 1-way ANOVA. (C) Flow cytometry quantification of mitophagy flux in β-cells from mt-Keima mice fed RFD or HFD for 15 weeks, followed by ex vivo exposure to DMSO or 1 μM C25-140 for 24 h. n = 3-4/group. ** P < 0.01 by 1-way ANOVA. (D) Flow cytometry quantification of mitophagy flux by MtPhagy dye approach in human β-cells following exposure to DMSO or 1 μM C25-140 for 24 h and exposed to GLT or control for 48 h. n = 4/group. * P < 0.05 by 1-way ANOVA. (E) WB with densitometry of phosphorylation of ubiquitin at serine 65 (pUb S65 ) in sgScram and sgTraf6 Min6 cells following exposure to BSA or 0.5 mM palmitate for 48 h and 250 nM valinomycin for the final 3 h. PPIB served as a loading control. n = 3/group. * P < 0.05 by 1-way ANOVA. (F) WB with densitometry of MFN2 in isolated islets from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks following 250 nM valinomycin exposure for up to 3 h. Densitometry represents relative change in MFN2 protein at 3 h following valinomycin exposure relative to 0 h baseline. PPIB served as a loading control. n = 3/group. *** P <0.001 by 1-way ANOVA. (G) WB with densitometry of LC3-II expression (ratio of LC3-II/(LC3-I+LC3-II)) in isolated islets from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. VCL served as an additional independent loading control. n = 3/group.

Journal: bioRxiv

Article Title: TRAF6 integrates innate immune signals to regulate glucose homeostasis via Parkin-dependent and -independent mitophagy

doi: 10.1101/2025.01.31.635900

Figure Lengend Snippet: (A) Differential RNA expression heatmap of selected mitochondrial biogenesis genes in isolated islets from control or Traf6 Δβ mice fed HFD for 15 weeks. n = 3-4/group. (B) Flow cytometry quantification of mitophagy flux by MtPhagy dye approach in β-cells from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. n = 5-6/group. *** P < 0.001 by 1-way ANOVA. (C) Flow cytometry quantification of mitophagy flux in β-cells from mt-Keima mice fed RFD or HFD for 15 weeks, followed by ex vivo exposure to DMSO or 1 μM C25-140 for 24 h. n = 3-4/group. ** P < 0.01 by 1-way ANOVA. (D) Flow cytometry quantification of mitophagy flux by MtPhagy dye approach in human β-cells following exposure to DMSO or 1 μM C25-140 for 24 h and exposed to GLT or control for 48 h. n = 4/group. * P < 0.05 by 1-way ANOVA. (E) WB with densitometry of phosphorylation of ubiquitin at serine 65 (pUb S65 ) in sgScram and sgTraf6 Min6 cells following exposure to BSA or 0.5 mM palmitate for 48 h and 250 nM valinomycin for the final 3 h. PPIB served as a loading control. n = 3/group. * P < 0.05 by 1-way ANOVA. (F) WB with densitometry of MFN2 in isolated islets from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks following 250 nM valinomycin exposure for up to 3 h. Densitometry represents relative change in MFN2 protein at 3 h following valinomycin exposure relative to 0 h baseline. PPIB served as a loading control. n = 3/group. *** P <0.001 by 1-way ANOVA. (G) WB with densitometry of LC3-II expression (ratio of LC3-II/(LC3-I+LC3-II)) in isolated islets from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. VCL served as an additional independent loading control. n = 3/group.

Techniques: RNA Expression, Isolation, Control, Flow Cytometry, Ex Vivo, Expressing

$(A) Schematic of proteomics sample preparation and experimental design. (B) Volcano plot of differentially expressed proteins in sgTraf6 palm vs. sgScram palm mitochondrial fractions. Differentially expressed proteins identified by adjusted P value < 0.05 and |log 2 FC| > 0.25. n = 4/group. (C) Workflow of protein candidate identification following proteomics analysis of mitochondrial fractions of sgTraf6 vs. sgScram Min6 cells following exposure to 0.5 mM palmitate or BSA for 48 h. (D) Differential protein expression heatmap of TAX1BP1, BNIP3, and p62, displayed as integrated mean log 2 FC, from proteomics studies of mitochondrial fractions of sgTraf6 vs. sgScram Min6 cells following exposure to 0.5 mM palmitate or BSA for 48 h. n = 4/group. (E) WB with densitometry of TAX1BP1, p62, and BNIP3 (relative to sgScram control) in mitochondrial fractions from sgScram and sgTraf6 Min6 cells exposed to BSA or 0.5 mM palmitate for 48 h. TOM20 served as a loading control. n = 3/group. * P < 0.05, ** P < 0.01, **** P < 0.0001 by 2-way ANOVA. (F) Representative deconvolution immunofluorescence images (left) from pancreatic sections of control or Traf6 Δβ mice fed RFD or HFD for 15 weeks stained for BNIP3 (green), mitochondria (SDHA; red), Insulin (cyan), and DNA (DAPI; blue). Insulin positive areas were selected and colocalization of BNIP3 and SDHA was quantified by Pearson’s correlation coefficient (right). n = 3/group. ** P < 0.01, *** P < 0.001 by 1-way ANOVA. Scale bar = 20 μm. (G) WB with densitometry of TAX1BP1, p62, and BNIP3 (relative to sgScram control) in whole cell lysates from sgScram and sgTraf6 Min6 cells exposed to BSA or 0.5 mM palmitate for 48 h. PPIB served as a loading control. n = 3/group. **** P < 0.0001 by 2-way ANOVA. (H) WB with densitometry of TAX1BP1, p62, and BNIP3 (relative to Control RFD) in islets isolated from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. PPIB served as a loading control. n = 3 per group. **** P < 0.0001 by 2-way ANOVA. ( I) Representative WB of FLAG, HA, and Myc levels following anti-FLAG or anti-IgG immunoprecipitation (IP) in Min6 cells transfected with plasmids expressing FLAG-TRAF6, HA-NRDP1, and Myc-Parkin and exposed to BSA or 0.5 mM palmitate for 48 h. VCL served as a loading control. n = 3/group. (J) WB with densitometry of NRDP1 and Parkin (relative to sgScram control) in mitochondrial fractions from sgScram and sgTraf6 Min6 cells exposed to BSA or 0.5 mM palmitate for 48 h. TOM20 served as a loading control. n = 3/group. ** P < 0.01, **** P < 0.0001 by 2-way ANOVA. (K) WB with densitometry of NRDP1 and Parkin (relative to sgScram control) in whole cell lysates of sgScram and sgTraf6 Min6 cells exposed to BSA or 0.5 mM palmitate for 48 h. VCL serves as a loading control. n = 3/group. *** P < 0.001 by 2-way ANOVA. (L) WB with densitometry of NRDP1 and Parkin (relative to Control RFD) in islets isolated from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. VCL served as a loading control. n = 3/ group. **** P < 0.0001 by 2-way ANOVA.$

Journal: bioRxiv

Article Title: TRAF6 integrates innate immune signals to regulate glucose homeostasis via Parkin-dependent and -independent mitophagy

doi: 10.1101/2025.01.31.635900

Figure Lengend Snippet: (A) Schematic of proteomics sample preparation and experimental design. (B) Volcano plot of differentially expressed proteins in sgTraf6 palm vs. sgScram palm mitochondrial fractions. Differentially expressed proteins identified by adjusted P value < 0.05 and |log 2 FC| > 0.25. n = 4/group. (C) Workflow of protein candidate identification following proteomics analysis of mitochondrial fractions of sgTraf6 vs. sgScram Min6 cells following exposure to 0.5 mM palmitate or BSA for 48 h. (D) Differential protein expression heatmap of TAX1BP1, BNIP3, and p62, displayed as integrated mean log 2 FC, from proteomics studies of mitochondrial fractions of sgTraf6 vs. sgScram Min6 cells following exposure to 0.5 mM palmitate or BSA for 48 h. n = 4/group. (E) WB with densitometry of TAX1BP1, p62, and BNIP3 (relative to sgScram control) in mitochondrial fractions from sgScram and sgTraf6 Min6 cells exposed to BSA or 0.5 mM palmitate for 48 h. TOM20 served as a loading control. n = 3/group. * P < 0.05, ** P < 0.01, **** P < 0.0001 by 2-way ANOVA. (F) Representative deconvolution immunofluorescence images (left) from pancreatic sections of control or Traf6 Δβ mice fed RFD or HFD for 15 weeks stained for BNIP3 (green), mitochondria (SDHA; red), Insulin (cyan), and DNA (DAPI; blue). Insulin positive areas were selected and colocalization of BNIP3 and SDHA was quantified by Pearson’s correlation coefficient (right). n = 3/group. ** P < 0.01, *** P < 0.001 by 1-way ANOVA. Scale bar = 20 μm. (G) WB with densitometry of TAX1BP1, p62, and BNIP3 (relative to sgScram control) in whole cell lysates from sgScram and sgTraf6 Min6 cells exposed to BSA or 0.5 mM palmitate for 48 h. PPIB served as a loading control. n = 3/group. **** P < 0.0001 by 2-way ANOVA. (H) WB with densitometry of TAX1BP1, p62, and BNIP3 (relative to Control RFD) in islets isolated from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. PPIB served as a loading control. n = 3 per group. **** P < 0.0001 by 2-way ANOVA. ( I) Representative WB of FLAG, HA, and Myc levels following anti-FLAG or anti-IgG immunoprecipitation (IP) in Min6 cells transfected with plasmids expressing FLAG-TRAF6, HA-NRDP1, and Myc-Parkin and exposed to BSA or 0.5 mM palmitate for 48 h. VCL served as a loading control. n = 3/group. (J) WB with densitometry of NRDP1 and Parkin (relative to sgScram control) in mitochondrial fractions from sgScram and sgTraf6 Min6 cells exposed to BSA or 0.5 mM palmitate for 48 h. TOM20 served as a loading control. n = 3/group. ** P < 0.01, **** P < 0.0001 by 2-way ANOVA. (K) WB with densitometry of NRDP1 and Parkin (relative to sgScram control) in whole cell lysates of sgScram and sgTraf6 Min6 cells exposed to BSA or 0.5 mM palmitate for 48 h. VCL serves as a loading control. n = 3/group. *** P < 0.001 by 2-way ANOVA. (L) WB with densitometry of NRDP1 and Parkin (relative to Control RFD) in islets isolated from control or Traf6 Δβ mice fed RFD or HFD for 15 weeks. VCL served as a loading control. n = 3/ group. **** P < 0.0001 by 2-way ANOVA.

Techniques: Sample Prep, Expressing, Control, Immunofluorescence, Staining, Isolation, Immunoprecipitation, Transfection

(A) Protein expression and densitometry quantification (relative to Control HFD) of TRAF6 and Parkin by WB in isolated islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. VCL served as a loading control. n = 4/group. **** P < 0.0001 by 1-way ANOVA. (B) Blood glucose concentrations measured during IPGTT (top) and AUC (bottom) of control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ male mice fed HFD for 15 weeks (left) or 30 weeks (right). n = 9-12/group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 2-way ANOVA (IPGTT) or 1-way ANOVA (AUC). (C) Serum insulin levels measured during in vivo glucose-stimulated insulin release assays in male mice fed HFD for 15 weeks. n = 8-10/group. ** P < 0.01 by 2-way ANOVA. (D) Glucose-stimulated insulin secretion from islets isolated from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 6-7/group. * P < 0.05, ** P < 0.01 by 2-way ANOVA. (E) KCl-stimulated insulin secretion from islets isolated from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 4/group. (F) Insulin content in islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 6-7/group. ( G) Pancreatic β-cell mass from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks (left) or 30 weeks (right). n = 4-5/group.

Journal: bioRxiv

Article Title: TRAF6 integrates innate immune signals to regulate glucose homeostasis via Parkin-dependent and -independent mitophagy

doi: 10.1101/2025.01.31.635900

Figure Lengend Snippet: (A) Protein expression and densitometry quantification (relative to Control HFD) of TRAF6 and Parkin by WB in isolated islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. VCL served as a loading control. n = 4/group. **** P < 0.0001 by 1-way ANOVA. (B) Blood glucose concentrations measured during IPGTT (top) and AUC (bottom) of control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ male mice fed HFD for 15 weeks (left) or 30 weeks (right). n = 9-12/group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 2-way ANOVA (IPGTT) or 1-way ANOVA (AUC). (C) Serum insulin levels measured during in vivo glucose-stimulated insulin release assays in male mice fed HFD for 15 weeks. n = 8-10/group. ** P < 0.01 by 2-way ANOVA. (D) Glucose-stimulated insulin secretion from islets isolated from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 6-7/group. * P < 0.05, ** P < 0.01 by 2-way ANOVA. (E) KCl-stimulated insulin secretion from islets isolated from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 4/group. (F) Insulin content in islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 6-7/group. ( G) Pancreatic β-cell mass from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks (left) or 30 weeks (right). n = 4-5/group.

Techniques: Expressing, Control, Isolation, In Vivo

$(A) Oxygen consumption rate (OCR) fractional change (left) and steady state quantification (right) following exposure to 3 mM glucose, 20 mM glucose, 10 μM oligomycin, and 3 mM KCN in isolated islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ islets isolated from mice fed HFD for 15 weeks. n = 3-6/group. ** P < 0.01 by 2-way ANOVA. (B) ATP/ADP ratio measured in isolated islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 4/group. ** P < 0.01, *** P < 0.001 by 2-way ANOVA. (C) mtDNA to nuclear DNA ratio in isolated islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 3-5/group. * P < 0.05, ** P < 0.01 by 1-way ANOVA. (D) Citrate synthase activity in isolated islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 3/group. * P < 0.05, ** P < 0.01 by 1-way ANOVA. (E) WB for OXPHOS subunits and TOM20 with densitometry (relative to Control HFD) in islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. VCL served as a loading control. n = 4/group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 2-way ANOVA. (F) Representative TEM images of β-cells from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. with quantification of mitochondrial morphology (∼100 independent mitochondria scored/animal). n = 3/group. * P < 0.05, ** P < 0.01, *** P < 0.001 by 1- way ANOVA. Scale bars: 500 nM. (G) Flow cytometry quantification of mitophagy flux by MtPhagy dye approach in β-cells from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 4-6/group. ** P < 0.01, *** P < 0.001 by 1-way ANOVA. ( H ) Representative deconvolution immunofluorescence images (left) from pancreatic sections of control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice HFD for 15 weeks stained for BNIP3 (green), mitochondria (SDHA; red), Insulin (cyan), and DNA (DAPI; blue). Insulin positive areas were selected and colocalization of BNIP3 and SDHA was quantified by Pearson’s correlation coefficient (right). n = 3/group. * P < 0.05, ** P < 0.01 by 1-way ANOVA. Scale bars: 20 μm.$

Journal: bioRxiv

Article Title: TRAF6 integrates innate immune signals to regulate glucose homeostasis via Parkin-dependent and -independent mitophagy

doi: 10.1101/2025.01.31.635900

Figure Lengend Snippet: (A) Oxygen consumption rate (OCR) fractional change (left) and steady state quantification (right) following exposure to 3 mM glucose, 20 mM glucose, 10 μM oligomycin, and 3 mM KCN in isolated islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ islets isolated from mice fed HFD for 15 weeks. n = 3-6/group. ** P < 0.01 by 2-way ANOVA. (B) ATP/ADP ratio measured in isolated islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 4/group. ** P < 0.01, *** P < 0.001 by 2-way ANOVA. (C) mtDNA to nuclear DNA ratio in isolated islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 3-5/group. * P < 0.05, ** P < 0.01 by 1-way ANOVA. (D) Citrate synthase activity in isolated islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 3/group. * P < 0.05, ** P < 0.01 by 1-way ANOVA. (E) WB for OXPHOS subunits and TOM20 with densitometry (relative to Control HFD) in islets from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. VCL served as a loading control. n = 4/group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 2-way ANOVA. (F) Representative TEM images of β-cells from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. with quantification of mitochondrial morphology (∼100 independent mitochondria scored/animal). n = 3/group. * P < 0.05, ** P < 0.01, *** P < 0.001 by 1- way ANOVA. Scale bars: 500 nM. (G) Flow cytometry quantification of mitophagy flux by MtPhagy dye approach in β-cells from control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice fed HFD for 15 weeks. n = 4-6/group. ** P < 0.01, *** P < 0.001 by 1-way ANOVA. ( H ) Representative deconvolution immunofluorescence images (left) from pancreatic sections of control, Traf6 Δβ , Parkin Δβ , and Traf6/Parkin Δβ mice HFD for 15 weeks stained for BNIP3 (green), mitochondria (SDHA; red), Insulin (cyan), and DNA (DAPI; blue). Insulin positive areas were selected and colocalization of BNIP3 and SDHA was quantified by Pearson’s correlation coefficient (right). n = 3/group. * P < 0.05, ** P < 0.01 by 1-way ANOVA. Scale bars: 20 μm.

Techniques: Isolation, Control, Activity Assay, Flow Cytometry, Immunofluorescence, Staining

Following exposure to TLR4 ligands or free fatty acids (FFA), TRAF6 interacts with ECSIT and NRDP1, and BNIP3 levels rise. TRAF6 localizes to the mitochondria where it complexes with Parkin and mediates the recruitment of ubiquitin receptors p62 and TAX1BP1. TRAF6 also restrains Parkin activity to support mitochondrial BNIP3 recruitment. Together, these events promote the induction of mitophagy to adaptively respond to metabolic stress and maintain β-cell function.

Journal: bioRxiv

Article Title: TRAF6 integrates innate immune signals to regulate glucose homeostasis via Parkin-dependent and -independent mitophagy

doi: 10.1101/2025.01.31.635900

Figure Lengend Snippet: Following exposure to TLR4 ligands or free fatty acids (FFA), TRAF6 interacts with ECSIT and NRDP1, and BNIP3 levels rise. TRAF6 localizes to the mitochondria where it complexes with Parkin and mediates the recruitment of ubiquitin receptors p62 and TAX1BP1. TRAF6 also restrains Parkin activity to support mitochondrial BNIP3 recruitment. Together, these events promote the induction of mitophagy to adaptively respond to metabolic stress and maintain β-cell function.

Techniques: Activity Assay, Cell Function Assay

Curcumin inhibits RANKL-promoted osteoclast-related protein expression in OCPs. (A) After the intervention with different concentration of curcumin (0, 5, 10 or 15 μM), the protein expression levels of TRAF6, NFATc1 and DC-stamp in OCPs were detected using Western-Blot assays. The samples came from the same experiment, and different gels were processed in parallel. (B–D) The histogram displays the relative quantitative results of protein levels in A . (E) With RANKL intervention, OCPs were treated with or without curcumin for the indicated times in α-MEM with 1% FBS (appropriate starvation for enhancing the phosphorylation effect). p-ERK, p-JNK and p-P38 were detected using Western-Blot assays. The expression of phosphorylated proteins is represented by the ratio of phosphorylated protein to total protein. The samples came from the same experiment, and different gels were processed in parallel. (F–H) The histogram displays the relative quantitative results of protein levels in E . (I–J) The lysates of corresponding OCPs were extracted with anti-TRAF6 or anti-RANK antibody for coimmunoprecipitation, and then the precipitation was detected by Western-Blot analyses with anti-RANK or anti-TRAF6 antibody, respectively. The samples came from the same experiment, and different gels were processed in parallel. The data are expressed as mean ± SEM from three independent experiments. ns, not significant; R, RANKL; CUR, curcumin; IP, the antibody for immunoprecipitation; IB, the antibody for immunoblot.

Journal: Biomedical Journal

Article Title: Curcumin suppresses RANKL-induced osteoclast precursor autophagy in osteoclastogenesis by inhibiting RANK signaling and downstream JNK-BCL2-Beclin1 pathway

doi: 10.1016/j.bj.2023.100605

Figure Lengend Snippet: Curcumin inhibits RANKL-promoted osteoclast-related protein expression in OCPs. (A) After the intervention with different concentration of curcumin (0, 5, 10 or 15 μM), the protein expression levels of TRAF6, NFATc1 and DC-stamp in OCPs were detected using Western-Blot assays. The samples came from the same experiment, and different gels were processed in parallel. (B–D) The histogram displays the relative quantitative results of protein levels in A . (E) With RANKL intervention, OCPs were treated with or without curcumin for the indicated times in α-MEM with 1% FBS (appropriate starvation for enhancing the phosphorylation effect). p-ERK, p-JNK and p-P38 were detected using Western-Blot assays. The expression of phosphorylated proteins is represented by the ratio of phosphorylated protein to total protein. The samples came from the same experiment, and different gels were processed in parallel. (F–H) The histogram displays the relative quantitative results of protein levels in E . (I–J) The lysates of corresponding OCPs were extracted with anti-TRAF6 or anti-RANK antibody for coimmunoprecipitation, and then the precipitation was detected by Western-Blot analyses with anti-RANK or anti-TRAF6 antibody, respectively. The samples came from the same experiment, and different gels were processed in parallel. The data are expressed as mean ± SEM from three independent experiments. ns, not significant; R, RANKL; CUR, curcumin; IP, the antibody for immunoprecipitation; IB, the antibody for immunoblot.

Article Snippet: Lentiviruses encoding TRAF6 cDNA or shRNA targeting TRAF6 (including the corresponding control vector) were constructed by homologous recombination between an expression vector (EX-Puro-Lv105) and cDNA or shRNA in 293 cells using construction kits (GeneCopoeia, MD, USA) in accordance with manufacturer's protocols.

Techniques: Expressing, Concentration Assay, Western Blot, Immunoprecipitation

Curcumin-inhibited autophagy is recovered by TRAF6 overexpression, and curcumin did not affect autophagy under TRAF6 silencing in OCPs. (A) After TRAF6 overexpression or TRAF6 silencing in OCPs treated with RANKL + M-CSF for 1 day, lentiviral transduction efficiency was identified using Western-Blot assays. (B) TRAF6-overexpressed OCPs continued to be induced using RANKL + M-CSF for 3 days with curcumin or PBS, and the formed TRAP + multinucleate cells in each group are regarded as differentiated osteoclasts. Scale bar, 50 μm. (C) The histogram represents the quantitative results of differentiated osteoclasts in B . (D) The histogram represents the quantitative results of large osteoclasts over 5 nuclei in B . (E) TRAF6-silenced OCPs continued to be induced using RANKL + M-CSF for 3 days with curcumin or PBS, and the formed TRAP + multinucleate cells in each group are regarded as differentiated osteoclasts. Scale bar, 50 μm. (F) The histogram represents the quantitative results of differentiated osteoclasts in E . (G) The histogram represents the quantitative results of large osteoclasts over 5 nuclei in E . (H) TRAF6-overexpressed or TRAF6-silenced OCPs continued to be induced by RANKL for 12 h with curcumin or PBS, and LC3 and p62 protein expression in OCPs were detected using Western-Blot assays. LC3 conversion rate was defined as the ratio of LC3II/LC3I. The samples came from the same experiment, and different gels were processed in parallel. The data are expressed as mean ± SEM from three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. ns, not significant; CUR, curcumin.

Journal: Biomedical Journal

Article Title: Curcumin suppresses RANKL-induced osteoclast precursor autophagy in osteoclastogenesis by inhibiting RANK signaling and downstream JNK-BCL2-Beclin1 pathway

doi: 10.1016/j.bj.2023.100605

Figure Lengend Snippet: Curcumin-inhibited autophagy is recovered by TRAF6 overexpression, and curcumin did not affect autophagy under TRAF6 silencing in OCPs. (A) After TRAF6 overexpression or TRAF6 silencing in OCPs treated with RANKL + M-CSF for 1 day, lentiviral transduction efficiency was identified using Western-Blot assays. (B) TRAF6-overexpressed OCPs continued to be induced using RANKL + M-CSF for 3 days with curcumin or PBS, and the formed TRAP + multinucleate cells in each group are regarded as differentiated osteoclasts. Scale bar, 50 μm. (C) The histogram represents the quantitative results of differentiated osteoclasts in B . (D) The histogram represents the quantitative results of large osteoclasts over 5 nuclei in B . (E) TRAF6-silenced OCPs continued to be induced using RANKL + M-CSF for 3 days with curcumin or PBS, and the formed TRAP + multinucleate cells in each group are regarded as differentiated osteoclasts. Scale bar, 50 μm. (F) The histogram represents the quantitative results of differentiated osteoclasts in E . (G) The histogram represents the quantitative results of large osteoclasts over 5 nuclei in E . (H) TRAF6-overexpressed or TRAF6-silenced OCPs continued to be induced by RANKL for 12 h with curcumin or PBS, and LC3 and p62 protein expression in OCPs were detected using Western-Blot assays. LC3 conversion rate was defined as the ratio of LC3II/LC3I. The samples came from the same experiment, and different gels were processed in parallel. The data are expressed as mean ± SEM from three independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. ns, not significant; CUR, curcumin.

Techniques: Over Expression, Transduction, Western Blot, Expressing

The enhanced osteoclast formation and OCP autophagy in Tg-hRANKL mice are reversed by curcumin. (A) Representative 3D micro-CT reconstructed images of the femurs from Tg-hRANKL mice fed with 200 mg/kg curcumin or the control diet (for 4 weeks; N = 6–10/group) and control mice in the same nest (WT mice) showing bone mass and bone microstructure (N = 6/group). Scale bar, 2 mm. (B) Representative H&E-stained tibial sections from each group. Scale bar, 20 μm. (C) Representative TRAP-stained tibial sections from each group (Black arrows indicate TRAP + cells). Scale bar, 5 μm. (D) Representative fluorescent images of TRAF6 in bone marrow RANK + CSF1R + cells sorted by FACS. Scale bar, 25 μm. (E) Representative TEM images of autophagosomes (red arrows) and autolysosomes (yellow arrows) in bone marrow RANK + CSF1R + cells. Scale bar, 2 μm. (F–L) The trabecular bone parameters, including BMD, BV/TV, BS/BV, Tb.Th, Tb.N, and Tb. Sp, were analysed via micro-CT. (M) The trabecular bone parameter, Tb.Ar, was analysed via H&E staining and IPP system. (N) The number of osteoclasts per millimeter of trabecular bone surface was counted. (O) The percentages of TRAF6-positive cells in E (30 cells per field, N = 5). (P) The quantitative results of autophagosomes and autolysosomes in F (75 cells from 3 independent experiments). The data are expressed as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. WT, control mice in the same nest; CUR, curcumin.

Journal: Biomedical Journal

Article Title: Curcumin suppresses RANKL-induced osteoclast precursor autophagy in osteoclastogenesis by inhibiting RANK signaling and downstream JNK-BCL2-Beclin1 pathway

doi: 10.1016/j.bj.2023.100605

Figure Lengend Snippet: The enhanced osteoclast formation and OCP autophagy in Tg-hRANKL mice are reversed by curcumin. (A) Representative 3D micro-CT reconstructed images of the femurs from Tg-hRANKL mice fed with 200 mg/kg curcumin or the control diet (for 4 weeks; N = 6–10/group) and control mice in the same nest (WT mice) showing bone mass and bone microstructure (N = 6/group). Scale bar, 2 mm. (B) Representative H&E-stained tibial sections from each group. Scale bar, 20 μm. (C) Representative TRAP-stained tibial sections from each group (Black arrows indicate TRAP + cells). Scale bar, 5 μm. (D) Representative fluorescent images of TRAF6 in bone marrow RANK + CSF1R + cells sorted by FACS. Scale bar, 25 μm. (E) Representative TEM images of autophagosomes (red arrows) and autolysosomes (yellow arrows) in bone marrow RANK + CSF1R + cells. Scale bar, 2 μm. (F–L) The trabecular bone parameters, including BMD, BV/TV, BS/BV, Tb.Th, Tb.N, and Tb. Sp, were analysed via micro-CT. (M) The trabecular bone parameter, Tb.Ar, was analysed via H&E staining and IPP system. (N) The number of osteoclasts per millimeter of trabecular bone surface was counted. (O) The percentages of TRAF6-positive cells in E (30 cells per field, N = 5). (P) The quantitative results of autophagosomes and autolysosomes in F (75 cells from 3 independent experiments). The data are expressed as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. WT, control mice in the same nest; CUR, curcumin.

Techniques: Micro-CT, Staining

The Working model diagram of this study. In brief, RANKL binds to its receptor RANK, and then activates the autophagy of OCPs through downstream TRAF6 signaling. The signal transduction downstream of TRAF6 is as follows: after phosphorylation by upstream signaling molecules, activated JNK leads to BCL2 phosphorylation at S70, thereby causing Beclin1 to dissociate from the BCL2-Beclin1 complex, which leads to Beclin1-dependent autophagy, and the differentiation of OCPs into mature osteoclasts. Furthermore, free BCL2 from the BCL2-Beclin1 complex binds to Bax, thereby inhibiting Bax-dependent apoptosis, which is also a promoter of osteoclast differentiation. Curcumin can inhibit the formation of mature osteoclasts by inhibiting RANKL-RANK-TRAF6-JNK-BCL2-Beclin1-autophagy activation signal transduction.

Journal: Biomedical Journal

Article Title: Curcumin suppresses RANKL-induced osteoclast precursor autophagy in osteoclastogenesis by inhibiting RANK signaling and downstream JNK-BCL2-Beclin1 pathway

doi: 10.1016/j.bj.2023.100605

Figure Lengend Snippet: The Working model diagram of this study. In brief, RANKL binds to its receptor RANK, and then activates the autophagy of OCPs through downstream TRAF6 signaling. The signal transduction downstream of TRAF6 is as follows: after phosphorylation by upstream signaling molecules, activated JNK leads to BCL2 phosphorylation at S70, thereby causing Beclin1 to dissociate from the BCL2-Beclin1 complex, which leads to Beclin1-dependent autophagy, and the differentiation of OCPs into mature osteoclasts. Furthermore, free BCL2 from the BCL2-Beclin1 complex binds to Bax, thereby inhibiting Bax-dependent apoptosis, which is also a promoter of osteoclast differentiation. Curcumin can inhibit the formation of mature osteoclasts by inhibiting RANKL-RANK-TRAF6-JNK-BCL2-Beclin1-autophagy activation signal transduction.

Techniques: Transduction, Activation Assay

Figure 4: TRAF6 is a direct downstream target of miR-146b-5p in HCC. A. The assumed miR-146b-5p binding sequences in the 3’-UTR of TRAF6. B. Wild-type miR-146b-5p crippled the luciferase activity that carried the 3’-UTR of TRAF6 mRNA. ***P<0.001. C-F. Alteration of miR-146b-5p levels regulated TRAF6 expression in vitro (C and D) and in vivo (E and F). The mRNA (C and E) and protein (D and F) expression levels of TRAF6 were down-regulated through miR-146b-5p over-expression and up-regulated by miR-146b- 5p knockdown. The mRNA levels were determined by qRT-PCR. The protein expression was detected by immunoblotting in vitro and IHC in vivo. **P<0.01. G. TRAF6 expression was lower in high miR-146b-5p group than that in low miR-146b-5p group. **P<0.01. All experiments were performed at least in triplicate and the data in B-G are presented as the (mean ± SD).

Journal: Oncotarget

Article Title: Down-regulation of miR-146b-5p by long noncoding RNA MALAT1 in hepatocellular carcinoma promotes cancer growth and metastasis.

doi: 10.18632/oncotarget.15640

Figure Lengend Snippet: Figure 4: TRAF6 is a direct downstream target of miR-146b-5p in HCC. A. The assumed miR-146b-5p binding sequences in the 3’-UTR of TRAF6. B. Wild-type miR-146b-5p crippled the luciferase activity that carried the 3’-UTR of TRAF6 mRNA. ***P<0.001. C-F. Alteration of miR-146b-5p levels regulated TRAF6 expression in vitro (C and D) and in vivo (E and F). The mRNA (C and E) and protein (D and F) expression levels of TRAF6 were down-regulated through miR-146b-5p over-expression and up-regulated by miR-146b- 5p knockdown. The mRNA levels were determined by qRT-PCR. The protein expression was detected by immunoblotting in vitro and IHC in vivo. **P<0.01. G. TRAF6 expression was lower in high miR-146b-5p group than that in low miR-146b-5p group. **P<0.01. All experiments were performed at least in triplicate and the data in B-G are presented as the (mean ± SD).

Article Snippet: TRAF6 cDNA clone vector (SC109844) was provided by OriGene Co. Ltd. (Beijing, China).

Techniques: Binding Assay, Luciferase, Activity Assay, Expressing, In Vitro, In Vivo, Over Expression, Knockdown, Quantitative RT-PCR, Western Blot

Figure 5: miR-146b-5p exerts its functions through inhibiting the TRAF6/p-Akt signaling pathway. In MHCC97-H cells with miR-146b-5p over-expression, the phosphorylation of Akt (p-Akt) was impaired and the expression levels of Bcl-2, Mcl-1 and MMP-9 were decreased. In Hep3B cells with miR-146b-5p knockdown, enhanced the phosphorylated Akt induced the expression of Bcl-2, Mcl-1 and MMP-9. Neither over-expression nor knockdown of miR-146b-5p could find any significant changes of total Akt (t-Akt) expression. *P<0.05. Immunoblotting was performed at least in triplicate and the data are presented as the (mean ± SD).

Journal: Oncotarget

Article Title: Down-regulation of miR-146b-5p by long noncoding RNA MALAT1 in hepatocellular carcinoma promotes cancer growth and metastasis.

doi: 10.18632/oncotarget.15640

Figure Lengend Snippet: Figure 5: miR-146b-5p exerts its functions through inhibiting the TRAF6/p-Akt signaling pathway. In MHCC97-H cells with miR-146b-5p over-expression, the phosphorylation of Akt (p-Akt) was impaired and the expression levels of Bcl-2, Mcl-1 and MMP-9 were decreased. In Hep3B cells with miR-146b-5p knockdown, enhanced the phosphorylated Akt induced the expression of Bcl-2, Mcl-1 and MMP-9. Neither over-expression nor knockdown of miR-146b-5p could find any significant changes of total Akt (t-Akt) expression. *P<0.05. Immunoblotting was performed at least in triplicate and the data are presented as the (mean ± SD).

Article Snippet: TRAF6 cDNA clone vector (SC109844) was provided by OriGene Co. Ltd. (Beijing, China).

Techniques: Over Expression, Phospho-proteomics, Expressing, Knockdown, Western Blot

Figure 6: Modification of TRAF6 expression partly abrogated the functions of miR-146b-5p on HCC cells. A-C. Modification of TRAF6 expression partly abolished the effects of miR-146b-5p on cell viability (A), apoptosis (B), migration and invasion (C) of MHHC97-H and Hep3B cells. *P<0.05, **P<0.01. D. TRAF6 abrogated the effects of miR-146b-5p for Akt phosphorylation as well as the protein levels of Bcl-2, Mcl-1 and MMP-9. **P<0.01. All experiments were performed at least in triplicate and the data in A-D are presented as the (mean ± SD).

Journal: Oncotarget

Article Title: Down-regulation of miR-146b-5p by long noncoding RNA MALAT1 in hepatocellular carcinoma promotes cancer growth and metastasis.

doi: 10.18632/oncotarget.15640

Figure Lengend Snippet: Figure 6: Modification of TRAF6 expression partly abrogated the functions of miR-146b-5p on HCC cells. A-C. Modification of TRAF6 expression partly abolished the effects of miR-146b-5p on cell viability (A), apoptosis (B), migration and invasion (C) of MHHC97-H and Hep3B cells. *P<0.05, **P<0.01. D. TRAF6 abrogated the effects of miR-146b-5p for Akt phosphorylation as well as the protein levels of Bcl-2, Mcl-1 and MMP-9. **P<0.01. All experiments were performed at least in triplicate and the data in A-D are presented as the (mean ± SD).

Article Snippet: TRAF6 cDNA clone vector (SC109844) was provided by OriGene Co. Ltd. (Beijing, China).

Techniques: Modification, Expressing, Migration, Phospho-proteomics

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