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Mimetics fndc5 knockout
Fndc5 Knockout, supplied by Mimetics, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 86 stars, based on 1 article reviews

fndc5 knockout - by Bioz Stars, 2026-05

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Myocardial infarction (MI) induces renal oxidative stress and activates the profibrotic TGF-β1/Smad2/3 signaling pathway, leading to interstitial collagen deposition and renal dysfunction. Resistance exercise upregulates renal FNDC5/Irisin expression. Irisin, potentially in concert with exercise-induced AMPK activation, attenuates oxidative stress and inhibits TGF-β1/Smad2/3 pathway activation, thereby reducing renal fibrosis and improving function. Solid lines indicate pathways supported by direct evidence from this study; dashed lines indicate proposed or associative links.

Journal: PLOS One

Article Title: FNDC5/Irisin-dependent renoprotection of resistance training in myocardial infarction–induced Type 2 cardiorenal syndrome

doi: 10.1371/journal.pone.0342468

Figure Lengend Snippet: Myocardial infarction (MI) induces renal oxidative stress and activates the profibrotic TGF-β1/Smad2/3 signaling pathway, leading to interstitial collagen deposition and renal dysfunction. Resistance exercise upregulates renal FNDC5/Irisin expression. Irisin, potentially in concert with exercise-induced AMPK activation, attenuates oxidative stress and inhibits TGF-β1/Smad2/3 pathway activation, thereby reducing renal fibrosis and improving function. Solid lines indicate pathways supported by direct evidence from this study; dashed lines indicate proposed or associative links.

Article Snippet: Eight-week-old healthy male C57BL/6J mice (purchased from the Animal Experiment Center of Xi’an Jiaotong University) and global FNDC5/Irisin knockout mice (customized by Cyagen Biosciences Inc.) were housed under standard laboratory conditions.

Techniques: Expressing, Activation Assay

a Schematic of MRI test and behavioral studies. b MRI scan of the brain in WT + TBI group and FNDC5-KO + TBI group; n = 6 for each group. c Quantification of lesion volume of MRI. d measurement of brain water content. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. WT + TBI group. e – g Several behavioral tests at different time points after TBI as measured by the grid-walking test ( e ), cylinder test ( f ) and adhesive removal test ( g ) ( n = 9 for each group). Significance was determined by Student’s t test ( c , d ) and two-way repeated ANOVA ( e – g ) with Bonferroni post hoc tests. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. WT+Sham group, # P < 0.05 and ## P < 0.01 vs. WT + TBI group. Values are presented as the mean ± SEM.

Journal: Cell Death & Disease

Article Title: FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control

doi: 10.1038/s41419-024-06748-w

Figure Lengend Snippet: a Schematic of MRI test and behavioral studies. b MRI scan of the brain in WT + TBI group and FNDC5-KO + TBI group; n = 6 for each group. c Quantification of lesion volume of MRI. d measurement of brain water content. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. WT + TBI group. e – g Several behavioral tests at different time points after TBI as measured by the grid-walking test ( e ), cylinder test ( f ) and adhesive removal test ( g ) ( n = 9 for each group). Significance was determined by Student’s t test ( c , d ) and two-way repeated ANOVA ( e – g ) with Bonferroni post hoc tests. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. WT+Sham group, # P < 0.05 and ## P < 0.01 vs. WT + TBI group. Values are presented as the mean ± SEM.

Article Snippet: FNDC5 knockout mice and Map2-CreERT2 mice were purchased from the Shanghai Model Organisms Center.

Techniques: Adhesive

a , b Representative DHE staining images and the quantitative results ( n = 6 for each group). Scale bar: 200 μm. c The effects of FNDC5 on MDA levels. d The effects of FNDC5 on mitochondrial MnSOD activity. e , f Representative images and statistical analysis of TUNEL staining of the perilesional cortex after TBI. Scale bar: 200 μm. g , h Representative Western blots and statistical analysis of the levels of Bax and Bcl-2 ( n = 6 for each group). Significance was determined by one-way ANOVA ( b – d , f , h ) with Bonferroni post hoc tests. * P < 0.05 and ** P < 0.01 vs. sham group, # P < 0.05 and ## P < 0.01 vs. TBI + NC group. Values are presented as the mean ± SEM.

Journal: Cell Death & Disease

Article Title: FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control

doi: 10.1038/s41419-024-06748-w

Figure Lengend Snippet: a , b Representative DHE staining images and the quantitative results ( n = 6 for each group). Scale bar: 200 μm. c The effects of FNDC5 on MDA levels. d The effects of FNDC5 on mitochondrial MnSOD activity. e , f Representative images and statistical analysis of TUNEL staining of the perilesional cortex after TBI. Scale bar: 200 μm. g , h Representative Western blots and statistical analysis of the levels of Bax and Bcl-2 ( n = 6 for each group). Significance was determined by one-way ANOVA ( b – d , f , h ) with Bonferroni post hoc tests. * P < 0.05 and ** P < 0.01 vs. sham group, # P < 0.05 and ## P < 0.01 vs. TBI + NC group. Values are presented as the mean ± SEM.

Article Snippet: FNDC5 knockout mice and Map2-CreERT2 mice were purchased from the Shanghai Model Organisms Center.

Techniques: Staining, Activity Assay, TUNEL Assay, Western Blot

a , b Western blot and statistical analysis of FNDC5 expression. c Representative MitoTracker fluorescence images illustrating mitochondrial morphology. Scale bar: 10 μm. d , e Mitochondrial morphological characteristics were quantified by ImageJ software. f ATP content of HT22 cells. g NAD + /NADH of HT22 cells. h , i Representative fluorescence intensity of JC-1 staining illustrating the MMP. Scale bar: 20 μm. j , k Representative MitoSOX fluorescence images of mitochondria-derived ROS. Scale bar: 50 μm ( n = 6 for each group). l , m Western blot and statistical analysis of SIRT3 expression. n Relative mRNA level of SIRT3. o Total acetylation level of mitochondrial proteins. p Relative acetylation level of mitochondrial proteins. Significance was determined by Student’s t test ( b , m , n , p ) and one-way ( d – g , i , k ) ANOVA with Bonferroni post hoc tests. * P < 0.05 and ** P < 0.01 vs. Control group, # P < 0.05 and ## P < 0.01 vs. FNDC5-KO + vel group. Values are presented as the mean ± SEM.

Journal: Cell Death & Disease

Article Title: FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control

doi: 10.1038/s41419-024-06748-w

Figure Lengend Snippet: a , b Western blot and statistical analysis of FNDC5 expression. c Representative MitoTracker fluorescence images illustrating mitochondrial morphology. Scale bar: 10 μm. d , e Mitochondrial morphological characteristics were quantified by ImageJ software. f ATP content of HT22 cells. g NAD + /NADH of HT22 cells. h , i Representative fluorescence intensity of JC-1 staining illustrating the MMP. Scale bar: 20 μm. j , k Representative MitoSOX fluorescence images of mitochondria-derived ROS. Scale bar: 50 μm ( n = 6 for each group). l , m Western blot and statistical analysis of SIRT3 expression. n Relative mRNA level of SIRT3. o Total acetylation level of mitochondrial proteins. p Relative acetylation level of mitochondrial proteins. Significance was determined by Student’s t test ( b , m , n , p ) and one-way ( d – g , i , k ) ANOVA with Bonferroni post hoc tests. * P < 0.05 and ** P < 0.01 vs. Control group, # P < 0.05 and ## P < 0.01 vs. FNDC5-KO + vel group. Values are presented as the mean ± SEM.

Article Snippet: FNDC5 knockout mice and Map2-CreERT2 mice were purchased from the Shanghai Model Organisms Center.

Techniques: Western Blot, Expressing, Fluorescence, Software, Staining, Derivative Assay, Control

a Representative DHE staining images in SIRT3 CKO mice and SIRT3 f/f mice. Scale bar: 200 μm. b The quantitative results of DHE staining images. c The effects of FNDC5 on MDA levels in SIRT3 CKO mice and in SIRT3 f/f mice. d The effects of FNDC5 on mitochondrial MnSOD activity in SIRT3 CKO mice and in SIRT3 f/f mice. e Representative images of TUNEL staining of the perilesional cortex 24 h in SIRT3 CKO mice and SIRT3 f/f mice. Scale bar: 200 μm. f , g Representative Western blot and statistical analysis of the levels of Bax and Bcl-2 in SIRT3 CKO mice and in SIRT3 f/f mice. ( n = 6 for each group). Significance was determined by two-way repeated ANOVA ( b , c , d , g ) with Bonferroni post hoc tests. * P < 0.05 and ** P < 0.01 vs. sham group, # P < 0.05 and ## P < 0.01 vs. TBI + NC group. Values are presented as the mean ± SEM.

Journal: Cell Death & Disease

Article Title: FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control

doi: 10.1038/s41419-024-06748-w

Figure Lengend Snippet: a Representative DHE staining images in SIRT3 CKO mice and SIRT3 f/f mice. Scale bar: 200 μm. b The quantitative results of DHE staining images. c The effects of FNDC5 on MDA levels in SIRT3 CKO mice and in SIRT3 f/f mice. d The effects of FNDC5 on mitochondrial MnSOD activity in SIRT3 CKO mice and in SIRT3 f/f mice. e Representative images of TUNEL staining of the perilesional cortex 24 h in SIRT3 CKO mice and SIRT3 f/f mice. Scale bar: 200 μm. f , g Representative Western blot and statistical analysis of the levels of Bax and Bcl-2 in SIRT3 CKO mice and in SIRT3 f/f mice. ( n = 6 for each group). Significance was determined by two-way repeated ANOVA ( b , c , d , g ) with Bonferroni post hoc tests. * P < 0.05 and ** P < 0.01 vs. sham group, # P < 0.05 and ## P < 0.01 vs. TBI + NC group. Values are presented as the mean ± SEM.

Article Snippet: FNDC5 knockout mice and Map2-CreERT2 mice were purchased from the Shanghai Model Organisms Center.

Techniques: Staining, Activity Assay, TUNEL Assay, Western Blot

a , b Western blot and statistical analysis of the levels of NRF2 expression in normal HT22 cell line and FNDC5 KO HT22 cell line. c Relative mRNA level of SIRT3 after treating with an NRF2 inhibitor (ML385). d , e Western blot and statistical analysis of the levels of SIRT3 expression. f FNDC5 deficiency does not affect the mRNA of NRF2. g The protein level of NRF2 was degraded faster over time in FNDC5 knockout cells after incubating with the mRNA synthesis inhibitor CHX. h statistical analysis of the levels of NRF2. i The protein level of NRF2 expression after incubating with the proteasome inhibitor MG132. j Statistical analysis of the levels of NRF2. k The protein level of NRF2 expression after incubating with the lysosomal inhibitor NH4Cl. l statistical analysis of the levels of NRF2. m Ubiquitination level of NRF2. Significance was determined by Student’s t test ( b , f ) and one-way ANOVA ( c , e ) or two-way repeated ANOVA ( h , j , l ) with Bonferroni post hoc tests. * P < 0.05 and ** P < 0.01 vs. Control group, # P < 0.05 and ## P < 0.01 vs. TBI + vector group, & P < 0.05 and && P < 0.01 vs. TBI + FNDC5 + veh group. Values are presented as the mean ± SEM.

Journal: Cell Death & Disease

Article Title: FNDC5 prevents oxidative stress and neuronal apoptosis after traumatic brain injury through SIRT3-dependent regulation of mitochondrial quality control

doi: 10.1038/s41419-024-06748-w

Figure Lengend Snippet: a , b Western blot and statistical analysis of the levels of NRF2 expression in normal HT22 cell line and FNDC5 KO HT22 cell line. c Relative mRNA level of SIRT3 after treating with an NRF2 inhibitor (ML385). d , e Western blot and statistical analysis of the levels of SIRT3 expression. f FNDC5 deficiency does not affect the mRNA of NRF2. g The protein level of NRF2 was degraded faster over time in FNDC5 knockout cells after incubating with the mRNA synthesis inhibitor CHX. h statistical analysis of the levels of NRF2. i The protein level of NRF2 expression after incubating with the proteasome inhibitor MG132. j Statistical analysis of the levels of NRF2. k The protein level of NRF2 expression after incubating with the lysosomal inhibitor NH4Cl. l statistical analysis of the levels of NRF2. m Ubiquitination level of NRF2. Significance was determined by Student’s t test ( b , f ) and one-way ANOVA ( c , e ) or two-way repeated ANOVA ( h , j , l ) with Bonferroni post hoc tests. * P < 0.05 and ** P < 0.01 vs. Control group, # P < 0.05 and ## P < 0.01 vs. TBI + vector group, & P < 0.05 and && P < 0.01 vs. TBI + FNDC5 + veh group. Values are presented as the mean ± SEM.

Article Snippet: FNDC5 knockout mice and Map2-CreERT2 mice were purchased from the Shanghai Model Organisms Center.

Techniques: Western Blot, Expressing, Knock-Out, Ubiquitin Proteomics, Control, Plasmid Preparation

Loss of Fndc5 attenuates the protection of NR against HFD-induced obesity and hepatic steatosis. (A) Generation of global Fndc5 knockout mice by targeting exon 2 of Fndc5 using CRISPR/CAS9 technology-mediated deletion. (B) Deletion of Fndc5 exon 2 was confirmed using DNA sequencing. The sequencing results showed that 984 base pairs in the Fndc5 nucleotide sequence were deleted. (C) Genotyping and the used primers are showed. WT, wild-type; MT, mutant. ( D ) Deletion of FNDC5 protein in liver and skeletal muscle tissue. (E) The body curves of WT and Fndc5 -/- mice fed with HFD and NR for 16 weeks . ** P <0.01, * P <0.05 vs Chow, ## P <0.01, # P <0.05 vs HFD, n = 6. (F-G) The liver weight (F) and the liver/body weight ratio (G) of WT and Fndc5 -/- mice fed with HFD and NR for 16 weeks. ** P <0.01 vs Chow, ## P <0.01 vs HFD, & P <0.05 Fndc5 -/- vs WT, n = 6. NS, no significance. (H-I) Hepatic cholesterol ( H ) and triglyceride ( I ) levels in liver of WT and Fndc5 -/- mice. * P <0.05 vs chow, ## P <0.01, # P <0.05 vs HFD, & P <0.05 Fndc5 -/- vs WT, n = 6-7. NS, no significance. (J) Oil Red O staining showing the lipid accumulation (red staining) in liver of mice. ** P <0.01 vs chow, ## P <0.01, # P <0.05 vs HFD, & P <0.05 Fndc5 -/- vs WT, n = 6. NS, no significance. n = 6-7.

Journal: Theranostics

Article Title: NAD + -boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine Fndc5/irisin

doi: 10.7150/thno.53652

Figure Lengend Snippet: Loss of Fndc5 attenuates the protection of NR against HFD-induced obesity and hepatic steatosis. (A) Generation of global Fndc5 knockout mice by targeting exon 2 of Fndc5 using CRISPR/CAS9 technology-mediated deletion. (B) Deletion of Fndc5 exon 2 was confirmed using DNA sequencing. The sequencing results showed that 984 base pairs in the Fndc5 nucleotide sequence were deleted. (C) Genotyping and the used primers are showed. WT, wild-type; MT, mutant. ( D ) Deletion of FNDC5 protein in liver and skeletal muscle tissue. (E) The body curves of WT and Fndc5 -/- mice fed with HFD and NR for 16 weeks . ** P <0.01, * P <0.05 vs Chow, ## P <0.01, # P <0.05 vs HFD, n = 6. (F-G) The liver weight (F) and the liver/body weight ratio (G) of WT and Fndc5 -/- mice fed with HFD and NR for 16 weeks. ** P <0.01 vs Chow, ## P <0.01 vs HFD, & P <0.05 Fndc5 -/- vs WT, n = 6. NS, no significance. (H-I) Hepatic cholesterol ( H ) and triglyceride ( I ) levels in liver of WT and Fndc5 -/- mice. * P <0.05 vs chow, ## P <0.01, # P <0.05 vs HFD, & P <0.05 Fndc5 -/- vs WT, n = 6-7. NS, no significance. (J) Oil Red O staining showing the lipid accumulation (red staining) in liver of mice. ** P <0.01 vs chow, ## P <0.01, # P <0.05 vs HFD, & P <0.05 Fndc5 -/- vs WT, n = 6. NS, no significance. n = 6-7.

Article Snippet: The global Fndc5 knockout mice (C57BL/6J-Fndc5 tm1cyagen , Fndc5 -/- , Serial Number: KOCMP-00230-Fndc5) in C57BL/6 genetic background were generated by Cyagen Bioscience Inc (Guangzhou, China) using CRISPR/Cas9 technology.

Techniques: Knock-Out, CRISPR, DNA Sequencing, Sequencing, Mutagenesis, Staining

Nicotinamide riboside, a well-established NAD+-boosting molecule, stimulates Fndc5/irisin in murine and human. (A-F) The influence of NR treatment (400 mg/kg/d) on plasma concentrations of FGF1, FGF21, resistin, and adiponectin in HFD-induced NAFLD mouse model. FGF1, FGF21, resistin and adiponectin plasma concentrations were changed in NAFLD mice but not by NR. ( E ) Plasma irisin concentration was changed by NR treatment. ** P <0.01 vs Chow, ## P <0.01 vs HFD, n = 8. NS, no significance. Two different commercial ELISA kits from Phoenix Pharmaceutical (Manufacturer P) and AdipoGen (Manufacturer A) were used. (F) The protein expression of Fndc5 in skeletal muscle of NAFLD mice with or without NR treatment. ** P <0.01 vs Chow, ## P <0.01 vs HFD, n = 4. (G) The protein expression of Fndc5 in other tissues such as liver and adipose of NAFLD mice with or without NR treatment. ** P <0.01 vs HFD by unpaired t -test, n = 4. (H-I) The influences of two-week NR supplement (500 mg, bid) or physical exercise on plasma irisin concentration in human volunteers detected by two different commercial ELISA kits from Phoenix Pharmaceutical (Manufacturer P, H ) and AdipoGen (Manufacturer A, I ). * P <0.05, ** P <0.01 by paired t -test, n = 6.

Journal: Theranostics

Article Title: NAD + -boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine Fndc5/irisin

doi: 10.7150/thno.53652

Figure Lengend Snippet: Nicotinamide riboside, a well-established NAD+-boosting molecule, stimulates Fndc5/irisin in murine and human. (A-F) The influence of NR treatment (400 mg/kg/d) on plasma concentrations of FGF1, FGF21, resistin, and adiponectin in HFD-induced NAFLD mouse model. FGF1, FGF21, resistin and adiponectin plasma concentrations were changed in NAFLD mice but not by NR. ( E ) Plasma irisin concentration was changed by NR treatment. ** P <0.01 vs Chow, ## P <0.01 vs HFD, n = 8. NS, no significance. Two different commercial ELISA kits from Phoenix Pharmaceutical (Manufacturer P) and AdipoGen (Manufacturer A) were used. (F) The protein expression of Fndc5 in skeletal muscle of NAFLD mice with or without NR treatment. ** P <0.01 vs Chow, ## P <0.01 vs HFD, n = 4. (G) The protein expression of Fndc5 in other tissues such as liver and adipose of NAFLD mice with or without NR treatment. ** P <0.01 vs HFD by unpaired t -test, n = 4. (H-I) The influences of two-week NR supplement (500 mg, bid) or physical exercise on plasma irisin concentration in human volunteers detected by two different commercial ELISA kits from Phoenix Pharmaceutical (Manufacturer P, H ) and AdipoGen (Manufacturer A, I ). * P <0.05, ** P <0.01 by paired t -test, n = 6.

Techniques: Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing

Loss of Fndc5 diminishes the improvement of NR against HFD-induced insulin resistance. (A-B) The ITT assay curve (A) and calculation of area under curve (AUC, B ) on WT and Fndc5 -/- mice. (C-D) The GTT assay curve (C) and calculation of AUC (D) on WT and Fndc5 -/- mice. (E-F) The phosphorylation of IRS-1 at tyrosine site 612 of IRS-1 (E) and serine sites at 307 and 636 sites of IRS-1 (F) in liver tissue of mice were detected by immunoblotting. (G) The phosphorylation of Akt at tyrosine site 308 in liver tissue of mice. ** P <0.01 vs chow, ## P <0.01, # P <0.05 vs HFD, & P <0.05 Fndc5 -/- vs WT, n = 6. NS, no significance.

Journal: Theranostics

Article Title: NAD + -boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine Fndc5/irisin

doi: 10.7150/thno.53652

Figure Lengend Snippet: Loss of Fndc5 diminishes the improvement of NR against HFD-induced insulin resistance. (A-B) The ITT assay curve (A) and calculation of area under curve (AUC, B ) on WT and Fndc5 -/- mice. (C-D) The GTT assay curve (C) and calculation of AUC (D) on WT and Fndc5 -/- mice. (E-F) The phosphorylation of IRS-1 at tyrosine site 612 of IRS-1 (E) and serine sites at 307 and 636 sites of IRS-1 (F) in liver tissue of mice were detected by immunoblotting. (G) The phosphorylation of Akt at tyrosine site 308 in liver tissue of mice. ** P <0.01 vs chow, ## P <0.01, # P <0.05 vs HFD, & P <0.05 Fndc5 -/- vs WT, n = 6. NS, no significance.

Techniques: Phospho-proteomics, Western Blot

Loss of Fndc5 counteracts the therapeutic efficacy of NR on HFD-induced hepatic inflammation and cell death. (A) F4/80 immunohistochemistry staining in liver tissue of WT and Fndc5 -/- mice. ( B ) CD11b immunohistochemistry staining in liver tissue of WT and Fndc5 -/- mice. ( C ) Tissue TNF-α, IL-6 and IL-1β protein levels in liver tissue of WT and Fndc5 -/- mice fed with HFD and NR. ( D ) The precursor and cleaved caspase-8 in liver tissue of WT and Fndc5 -/- mice. ( E ) TUNEL staining in liver tissue of WT and Fndc5 -/- mice. ** P <0.01 vs chow, # P <0.05, ## P <0.01 vs HFD, & P <0.05 Fndc5 -/- vs WT, n = 6. NS, no significance.

Journal: Theranostics

Article Title: NAD + -boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine Fndc5/irisin

doi: 10.7150/thno.53652

Figure Lengend Snippet: Loss of Fndc5 counteracts the therapeutic efficacy of NR on HFD-induced hepatic inflammation and cell death. (A) F4/80 immunohistochemistry staining in liver tissue of WT and Fndc5 -/- mice. ( B ) CD11b immunohistochemistry staining in liver tissue of WT and Fndc5 -/- mice. ( C ) Tissue TNF-α, IL-6 and IL-1β protein levels in liver tissue of WT and Fndc5 -/- mice fed with HFD and NR. ( D ) The precursor and cleaved caspase-8 in liver tissue of WT and Fndc5 -/- mice. ( E ) TUNEL staining in liver tissue of WT and Fndc5 -/- mice. ** P <0.01 vs chow, # P <0.05, ## P <0.01 vs HFD, & P <0.05 Fndc5 -/- vs WT, n = 6. NS, no significance.

Techniques: Drug discovery, Immunohistochemistry, Staining, TUNEL Assay

Loss of Fndc5 counteracts the therapeutic efficacy of NR on HFD-induced liver fibrosis and injury. (A) α-SMA staining in liver tissue of WT and Fndc5 -/- mice. (B) Masson's staining in liver tissue of WT and Fndc5 -/- mice. (C) qPCR analysis showing the TGF-β and TIMP-1 mRNA in liver tissue of WT and Fndc5 -/- mice. (D) Immunoblotting analysis showing the HMGB-1 protein expression in liver tissue of WT and Fndc5 -/- mice. (E-G) Plasma activities of AST (E) , ALT (F) and ALP (G) in WT and Fndc5 -/- mice. ** P <0.01 vs chow, # P <0.05, ## P <0.01 vs HFD, & P <0.05 Fndc5 -/- vs WT, n = 6. NS, no significance.

Journal: Theranostics

Article Title: NAD + -boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine Fndc5/irisin

doi: 10.7150/thno.53652

Figure Lengend Snippet: Loss of Fndc5 counteracts the therapeutic efficacy of NR on HFD-induced liver fibrosis and injury. (A) α-SMA staining in liver tissue of WT and Fndc5 -/- mice. (B) Masson's staining in liver tissue of WT and Fndc5 -/- mice. (C) qPCR analysis showing the TGF-β and TIMP-1 mRNA in liver tissue of WT and Fndc5 -/- mice. (D) Immunoblotting analysis showing the HMGB-1 protein expression in liver tissue of WT and Fndc5 -/- mice. (E-G) Plasma activities of AST (E) , ALT (F) and ALP (G) in WT and Fndc5 -/- mice. ** P <0.01 vs chow, # P <0.05, ## P <0.01 vs HFD, & P <0.05 Fndc5 -/- vs WT, n = 6. NS, no significance.

Techniques: Drug discovery, Staining, Western Blot, Expressing, Clinical Proteomics

Loss of Fndc5 revokes the beneficial effects of NR on genes involved in mitochondrial biogenesis and mitophagy in NAFLD mice. ( A-G ) The mRNA expression of TFMA (A) , NRF-1 (B) , PGC-1α (C) , Mfn2 (D) , Mst1 (E) , NR4A1 (F) and Bnip4 (G) in liver tissue of WT and FNDC5 -/- mice was determined using quantitative PCR analysis. (H-J) Mitochondria were isolated and the activities of Complex I, II and IV in mitochondrial extracts were determined. (K) The acetylation of PGC-1α in liver tissue of WT and FNDC5 -/- mice was measured. The liver samples were immunoprecipitated with a monoclonal against PGC-1α and then probed by an antibody against acetylated-lysine with immunoblotting. (L) The NAD + levels in liver tissue of WT and FNDC5 -/- mice. * P <0.05 vs chow; # P <0.05, ## P <0.01 vs HFD; & P <0.01 Fndc -/- vs WT, n = 6. NS, no significance.

Journal: Theranostics

Article Title: NAD + -boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine Fndc5/irisin

doi: 10.7150/thno.53652

Figure Lengend Snippet: Loss of Fndc5 revokes the beneficial effects of NR on genes involved in mitochondrial biogenesis and mitophagy in NAFLD mice. ( A-G ) The mRNA expression of TFMA (A) , NRF-1 (B) , PGC-1α (C) , Mfn2 (D) , Mst1 (E) , NR4A1 (F) and Bnip4 (G) in liver tissue of WT and FNDC5 -/- mice was determined using quantitative PCR analysis. (H-J) Mitochondria were isolated and the activities of Complex I, II and IV in mitochondrial extracts were determined. (K) The acetylation of PGC-1α in liver tissue of WT and FNDC5 -/- mice was measured. The liver samples were immunoprecipitated with a monoclonal against PGC-1α and then probed by an antibody against acetylated-lysine with immunoblotting. (L) The NAD + levels in liver tissue of WT and FNDC5 -/- mice. * P <0.05 vs chow; # P <0.05, ## P <0.01 vs HFD; & P <0.01 Fndc -/- vs WT, n = 6. NS, no significance.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Immunoprecipitation, Western Blot

NR inhibits ubiquitination of Fndc5 to stabilize Fndc5. (A-B) Fndc5 mRNA expression in mouse liver tissue (A) and AML12 hepatocytes (B) upon NR administration. n = 5-7. NS, no significance. (C) Evaluation of Fndc5 protein degradation and stability under vehicle control (saline) or NR treatment (300 µM) using cycloheximide (Chx, 3 µM) incubation in AML12 hepatocytes. * P <0.05, ** P <0.01 vs control AML12 cells. n = 4. (D) Influence of lactacystin, MG132, bafilomycin A1 (Baf A1) and chloroquine (CQ) on Fndc5 protein expression. Tubulin was used as a loading control. (E) Effects of proteasome inhibitors (bortezomib and MG132) and autophagy inhibitor Baf-A1 on Fndc5 ubiquitination in cultured AML12 hepatocytes. The cell extracts were immunoprecipitated with anti-flag antibody and then probed by anti-ubiquitin and anti-Fndc5 antibodies. (F) Ubiquitination of Fndc5 under palmitic acid (PA) or NR. MG132 was added to block ubiquitin-proteasome degradation system. The cell extracts were immunoprecipitated with an anti-flag antibody and then probed by anti-ubiquitin and anti-Fndc5 antibodies. ** P <0.01 vs without PA. ## P <0.01 vs PA. n = 4. IP, immunoprecipitation. WB, western blotting.

Journal: Theranostics

Article Title: NAD + -boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine Fndc5/irisin

doi: 10.7150/thno.53652

Figure Lengend Snippet: NR inhibits ubiquitination of Fndc5 to stabilize Fndc5. (A-B) Fndc5 mRNA expression in mouse liver tissue (A) and AML12 hepatocytes (B) upon NR administration. n = 5-7. NS, no significance. (C) Evaluation of Fndc5 protein degradation and stability under vehicle control (saline) or NR treatment (300 µM) using cycloheximide (Chx, 3 µM) incubation in AML12 hepatocytes. * P <0.05, ** P <0.01 vs control AML12 cells. n = 4. (D) Influence of lactacystin, MG132, bafilomycin A1 (Baf A1) and chloroquine (CQ) on Fndc5 protein expression. Tubulin was used as a loading control. (E) Effects of proteasome inhibitors (bortezomib and MG132) and autophagy inhibitor Baf-A1 on Fndc5 ubiquitination in cultured AML12 hepatocytes. The cell extracts were immunoprecipitated with anti-flag antibody and then probed by anti-ubiquitin and anti-Fndc5 antibodies. (F) Ubiquitination of Fndc5 under palmitic acid (PA) or NR. MG132 was added to block ubiquitin-proteasome degradation system. The cell extracts were immunoprecipitated with an anti-flag antibody and then probed by anti-ubiquitin and anti-Fndc5 antibodies. ** P <0.01 vs without PA. ## P <0.01 vs PA. n = 4. IP, immunoprecipitation. WB, western blotting.

Techniques: Ubiquitin Proteomics, Expressing, Control, Saline, Incubation, Cell Culture, Immunoprecipitation, Blocking Assay, Western Blot

SIRT2 interacts with Fndc5 to promote its deacetylation and deubiquitination. (A) Inhibition of deacetylases by DIC abolishes the action of NR on Fndc5 deubiquitination in HepG2 cells. DIC, deacetylation inhibition cocktail (100×). The extracts of cells treated as indicated were immunoprecipitated with anti-flag antibody and then probed by anti-ubiquitin and anti-Fndc5 antibodies. ** P <0.01 PA vs veh, # P <0.05 NR vs PA; n = 3. NS, no significance. (B) Effects of siRNA-mediated SIRT1-7 knockdown on Fndc5 protein expression in HepG2 cells under PA and NR treatment. ** P <0.01 NR vs PA; # P <0.05, ## P <0.01 vs PA + NR; n = 3. NS, no significance. (C) Flag-Fndc5 and myc-SIRT2 were co-transfected into HepG2 cells to detect the interaction between them using co-immunoprecipitation assay. (D) The interaction between endogenous Fndc5 and SIRT2 was detected using immunoprecipitation and Western blotting assay. Normal IgG was used as a normal control. (E) The influences of NR treatment or SIRT2 overexpression on Fndc5 acetylation were detected. The Flag-Fndc5-transfected cells treated as indicated were lysed and immunoprecipitated with an anti-flag antibody, and then probed by an anti-Ac-lys antibody. ( F ) The ubiquitination of Fndc5 in control and SIRT2-knockdown cells.

Journal: Theranostics

Article Title: NAD + -boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine Fndc5/irisin

doi: 10.7150/thno.53652

Figure Lengend Snippet: SIRT2 interacts with Fndc5 to promote its deacetylation and deubiquitination. (A) Inhibition of deacetylases by DIC abolishes the action of NR on Fndc5 deubiquitination in HepG2 cells. DIC, deacetylation inhibition cocktail (100×). The extracts of cells treated as indicated were immunoprecipitated with anti-flag antibody and then probed by anti-ubiquitin and anti-Fndc5 antibodies. ** P <0.01 PA vs veh, # P <0.05 NR vs PA; n = 3. NS, no significance. (B) Effects of siRNA-mediated SIRT1-7 knockdown on Fndc5 protein expression in HepG2 cells under PA and NR treatment. ** P <0.01 NR vs PA; # P <0.05, ## P <0.01 vs PA + NR; n = 3. NS, no significance. (C) Flag-Fndc5 and myc-SIRT2 were co-transfected into HepG2 cells to detect the interaction between them using co-immunoprecipitation assay. (D) The interaction between endogenous Fndc5 and SIRT2 was detected using immunoprecipitation and Western blotting assay. Normal IgG was used as a normal control. (E) The influences of NR treatment or SIRT2 overexpression on Fndc5 acetylation were detected. The Flag-Fndc5-transfected cells treated as indicated were lysed and immunoprecipitated with an anti-flag antibody, and then probed by an anti-Ac-lys antibody. ( F ) The ubiquitination of Fndc5 in control and SIRT2-knockdown cells.

Techniques: Inhibition, Immunoprecipitation, Ubiquitin Proteomics, Knockdown, Expressing, Transfection, Co-Immunoprecipitation Assay, Western Blot, Control, Over Expression

Identification of key lysine sites for SIRT2-mediated Fndc5 deubiquitination and deacetylation. (A) Comparison of Fndc5 protein sequences among mouse, rat, cattle, dog, cat, oreochromis niloticus, zebrafish and human species. The identical sites were highlighted by red and the conservative lysine sites were indicated by asterisks. (B) Plasmids carrying wild-type (WT) and mutant Fndc5 (MT) were generated. The K127 and K131 sites in MT1-Fndc5, K177 site in MT2-Fndc5 and K185, K187 and K189 sites in MT3-Fndc5 were mutated into arginine (R). (C) WT, MT1-Fndc5, MT2-Fndc5 and MT3-Fndc5 were transfected into HepG2 cell line respectively and then treated with PA and NR. To monitor the ubiquitination of these Fndc5 proteins, the cells were lysed and the extracts were immunoprecipitated by anti-Flag antibody followed by immunoblotting with anti-Ubiquitin. ** P <0.01 vs WT-Fndc5. N = 3. (D) Cells were transfected and treated as in (C) and the cell extracts were immunoprecipitated by anti-Flag antibody followed by immunoblotting with anti-Acetylated-lysine (anti-Ac) to monitor the acetylation of Fndc5 proteins. ** P <0.01 vs WT Fndc5. N = 3. (E) The Fndc5 protein expression in HepG2 cells transfected with plasmids carrying WT and mutant Fndc5 was determined. ** P <0.01 vs Mock. ## P <0.01 vs WT-Fndc5. N = 3.

Journal: Theranostics

Article Title: NAD + -boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine Fndc5/irisin

doi: 10.7150/thno.53652

Figure Lengend Snippet: Identification of key lysine sites for SIRT2-mediated Fndc5 deubiquitination and deacetylation. (A) Comparison of Fndc5 protein sequences among mouse, rat, cattle, dog, cat, oreochromis niloticus, zebrafish and human species. The identical sites were highlighted by red and the conservative lysine sites were indicated by asterisks. (B) Plasmids carrying wild-type (WT) and mutant Fndc5 (MT) were generated. The K127 and K131 sites in MT1-Fndc5, K177 site in MT2-Fndc5 and K185, K187 and K189 sites in MT3-Fndc5 were mutated into arginine (R). (C) WT, MT1-Fndc5, MT2-Fndc5 and MT3-Fndc5 were transfected into HepG2 cell line respectively and then treated with PA and NR. To monitor the ubiquitination of these Fndc5 proteins, the cells were lysed and the extracts were immunoprecipitated by anti-Flag antibody followed by immunoblotting with anti-Ubiquitin. ** P <0.01 vs WT-Fndc5. N = 3. (D) Cells were transfected and treated as in (C) and the cell extracts were immunoprecipitated by anti-Flag antibody followed by immunoblotting with anti-Acetylated-lysine (anti-Ac) to monitor the acetylation of Fndc5 proteins. ** P <0.01 vs WT Fndc5. N = 3. (E) The Fndc5 protein expression in HepG2 cells transfected with plasmids carrying WT and mutant Fndc5 was determined. ** P <0.01 vs Mock. ## P <0.01 vs WT-Fndc5. N = 3.

Techniques: Comparison, Mutagenesis, Generated, Transfection, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Expressing

Blockade of SIRT2 by AGK2 compromises the therapeutic action of NR against NAFLD. (A-E) Liver SIRT2 activity (A) , liver NAD + level (B) , plasma NAD + level (C) , Serum ALT activity (D) and serum irisin level were determined in five groups of mice. NAFLD model was induced by MCD diet for 4 weeks. NR (400 mg/kg/day) and AGK2 (1 mg/kg/day) were injected intraperitoneally. * P < 0.05, ** P < 0.01 vs Chow. # P < 0.05, ## P < 0.01 vs NAFLD. & P < 0.05, && P < 0.01 vs NAFLD + NR. N = 6 per group. (F-I) Representative images and quantitative analysis of lipid accumulation, NAFLD activity score, macrophage infiltration and liver fibrosis according to Oil Red O staining, H & E staining, F4/80 immunohistochemistry staining and Masson trichrome staining respectively. * P < 0.05, ** P < 0.01 vs NAFLD. # P < 0.05, ## P < 0.01 vs NAFLD + NR. N = 6 per group. (J-K) Fndc5 protein expression in mice under normal (J) or NAFLD (K) status. (L-M) The liver tissues were lysed and then immunoprecipitated by anti-Fndc5 antibody followed by immunoblotting with anti-Acetylated-lysine (anti-Ac, L ) or anti-Ubiquitin (M) to monitor the acetylation or ubiquitination of endogenous Fndc5 in mice treated with NR or NR plus AGK2. AGK2 treatment significantly abolished the decreased acetylation and ubiquitination of Fndc5 by NR.

Journal: Theranostics

Article Title: NAD + -boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine Fndc5/irisin

doi: 10.7150/thno.53652

Figure Lengend Snippet: Blockade of SIRT2 by AGK2 compromises the therapeutic action of NR against NAFLD. (A-E) Liver SIRT2 activity (A) , liver NAD + level (B) , plasma NAD + level (C) , Serum ALT activity (D) and serum irisin level were determined in five groups of mice. NAFLD model was induced by MCD diet for 4 weeks. NR (400 mg/kg/day) and AGK2 (1 mg/kg/day) were injected intraperitoneally. * P < 0.05, ** P < 0.01 vs Chow. # P < 0.05, ## P < 0.01 vs NAFLD. & P < 0.05, && P < 0.01 vs NAFLD + NR. N = 6 per group. (F-I) Representative images and quantitative analysis of lipid accumulation, NAFLD activity score, macrophage infiltration and liver fibrosis according to Oil Red O staining, H & E staining, F4/80 immunohistochemistry staining and Masson trichrome staining respectively. * P < 0.05, ** P < 0.01 vs NAFLD. # P < 0.05, ## P < 0.01 vs NAFLD + NR. N = 6 per group. (J-K) Fndc5 protein expression in mice under normal (J) or NAFLD (K) status. (L-M) The liver tissues were lysed and then immunoprecipitated by anti-Fndc5 antibody followed by immunoblotting with anti-Acetylated-lysine (anti-Ac, L ) or anti-Ubiquitin (M) to monitor the acetylation or ubiquitination of endogenous Fndc5 in mice treated with NR or NR plus AGK2. AGK2 treatment significantly abolished the decreased acetylation and ubiquitination of Fndc5 by NR.

Techniques: Activity Assay, Clinical Proteomics, Injection, Staining, Immunohistochemistry, Expressing, Immunoprecipitation, Western Blot, Ubiquitin Proteomics

A proposed framework for the involvement of Fndc5/irisin in the protection of NR against NAFLD.

Journal: Theranostics

Article Title: NAD + -boosting therapy alleviates nonalcoholic fatty liver disease via stimulating a novel exerkine Fndc5/irisin

doi: 10.7150/thno.53652

Figure Lengend Snippet: A proposed framework for the involvement of Fndc5/irisin in the protection of NR against NAFLD.

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