Journal: Cell reports

Article Title: JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

doi: 10.1016/j.celrep.2016.08.006

Figure Lengend Snippet: (A) Immortalized normal diploid human fibroblast cells containing a chromosomally integrated NHEJ reporter cassette (see ) were co-transfected with I-SceI and DsRed expression vectors as well as either an SIRT6-encoding plasmid or a control plasmid in the presence or absence of paraquat and a JNK inhibitor (SP600125). SIRT6 expression stimulated NHEJ 2.3-fold relative to control; when cells were pretreated with 1 mM paraquat, SIRT6 expression stimulated NHEJ 9.4-fold relative to control. Pretreating cells with 10 µMJNK inhibitor did not affect the ability of SIRT6 to stimulate NHEJ under basal conditions; when cells were pretreated with both paraquat and JNK inhibitor, however, SIRT6 failed to stimulate NHEJ. Western blots indicate activation of JNK signaling in response to paraquat, as indicated by phosphorylation of c-JUN (p-cJUN); treatment with the JNK inhibitor SP600125 effectively abrogated JNK signaling, but did not affect the paraquat-induced increase in the levels of SIRT6 protein (bottom panel). Error bars indicate SD (n = 6). See also . (B) The requirement of JNK signaling for SIRT6 expression to stimulate NHEJ in response to stress was confirmed using siRNAs. HCA2-hTERT-NHEJ cells were transfected as in (A), but, instead of exposure to a chemical inhibitor, the cells were co-transfected with siRNAs specific to JNK1/2 or a scrambled, control siRNA. SIRT6 expression massively stimulated NHEJ in cells pretreated with paraquat, but failed to do so when the cells also had been transfected with JNK siRNAs. Western blots indicate activation of JNK signaling in response to paraquat, as indicated by phosphorylation of c-JUN (p-cJUN); treatment siRNAs targeting JNK effectively abrogated JNK signaling. Error bars indicate SD (n = 5). (C) Immortalized normal diploid human fibroblast cells containing a chromosomally integrated HR reporter cassette (see ) were co-transfected with I-SceI and DsRed expression vectors as well as either an SIRT6-encoding plasmid or a control plasmid in the presence or absence of paraquat and a JNK inhibitor (SP600125). SIRT6 expression stimulated HR 3.1-fold relative to control; when cells were pretreated with 1 mM paraquat, SIRT6 expression stimulated NHEJ 10.4-fold relative to control. Pretreating cells with 10 µM JNK inhibitor did not affect the ability of SIRT6 to stimulate HR under basal conditions; when cells were pretreated with both paraquat and JNK inhibitor, however, SIRT6 failed to stimulate HR. Error bars indicate SD (n = 4). (D) SIRT6 expression accelerates the clearance of the DNA DSB marker γH2AX in HCA2-hTERT cells that had been pretreated with 1 mM paraquat for 16 hr. Inhibition of JNK signaling with SP600125 or JNK siRNA abrogates the effect of SIRT6 overexpression. Data represent the average number of γH2AX foci per nucleus. At least 50 nuclei were scored for each time point. Error bars indicate SEM. (E) Human fibroblasts, transfected with a plasmid encoding either SIRT6 or a control vector, were treated with 1 mM paraquat for 16 hr. Repair was measured 3 hr after the treatment using a comet assay kit (Trevigen) according to the manufacturer’s instructions. Tail moments were determined using CometScore software. One hundred cells were scored for each independent experiment. Error bars indicate SD (n = 3; *p < 0.05 and **p < 0.01). See also for inhibitors of other kinases.

Article Snippet: Purified, anisomycin-activated JNK (1 μg, Signal Chem, M34-10G) was incubated with 5 μg bacterially purified recombinant SIRT6 for 2 hr in Kinase Reaction Buffers (Signal Chem, K23-09-05 and K21-09-05) and ATP or [γ- 32 P]-ATP.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Western Blot, Activation Assay, Phospho-proteomics, Marker, Inhibition, Over Expression, Single Cell Gel Electrophoresis, Software

Journal: Cell reports

Article Title: JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

doi: 10.1016/j.celrep.2016.08.006

Figure Lengend Snippet: (A) CoIP reveals that SIRT6 interacts with JNK in HCA2-hTERT cells only when the cells have been exposed to oxidative stress (1 mM paraquat for 16hr). The experiment was repeated at least four times. (B) In vitro phosphorylation assay demonstrating that JNK can phosphorylate SIRT6 in vitro. Anisomysin-activated JNK, purified from HEK293 cells, was incubated with BSA and bacterially purified SIRT6 in the presence of 32 P-ATP and a kinase reaction buffer. SIRT6 specifically incorporated the radiolabel in these reactions, indicating that it was phosphorylated by JNK. The experiment was repeated three times and a representative gel is shown. See also . (C) SIRT6 plasmids encoding mutations at the indicated putative phosphorylation sites were overexpressed in HCA2-hTERT-NHEJ cells to measure their ability to stimulate NHEJ. SIRT6 T294A, S303A, S330A, and S338A all stimulated NHEJ similarly to WT SIRT6. SIRT6 S10A, however, failed to stimulate NHEJ in response to stress. Expression of an SIRT6 plasmid encoding an S10E phospho-mimetic mutation was able to powerfully stimulate NHEJ in the absence of oxidative stress. The effect of S10E mutation on DNA repair was resistant to JNK inhibition with SP600125. Error bars indicate SD (n = 4). Immunoblot (above) demonstrates that all of the indicated SIRT6 vectors were expressed stably and at comparable levels (*p < 0.05 and **p < 0.01). (D) NHEJ reporter construct was integrated into SIRT6 −/− MEF to measure the SIRT6 S10A and S10E activity in DNA repair in the absence of endogenous SIRT6. Cells expressing SIRT6 S10A mutant showed no stimulation of NHEJ repair in response to paraquat-induced oxidative stress. SIRT6 S10E phospho-mimetic mutant stimulated NHEJ under basal conditions, which could be further stimulated by stress; however, this additional stimulation of NHEJ was not affected by JNK inhibitor SP600125. Error bars indicate SD (n = 3; *p < 0.05 and **p < 0.01). (E) In vitro phosphorylation assay demonstrates that, while JNK can phosphorylate WT SIRT6, it cannot phosphorylate SIRT6 S10A. Anisomysin-activated JNK, purified fromHEK293 cells, was incubated with BSA and bacterially purified WT SIRT6 of SIRT6 S10A in the presence of 32 P-ATP and a kinase reaction buffer. The experiment was repeated three times and a representative gel is shown. (F) SIRT6 is phosphorylated on S10 in vivo after oxidative stress and the phosphorylation is diminished by JNK inhibitor (SP600125). Custom rabbit polyclonal antibodies (Rb5159 and Rb5160) were generated by immunizing rabbits with YAAGL pS PYADKGKC peptide (see for antibody specificity assays). The hTERT-immortalized human fibroblasts HCA2 were transfected with WT SIRT6-expressing plasmid, then treated with paraquat and/or JNK inhibitor and SIRT6 S10-P, and total SIRT6 levels were assessed by western blot (Rb5159 is pictured; both antibodies gave comparable results). To further confirm the specificity of S10-P antibodies, replicate samples were run and treated with Lambda Protein Phosphatase (LPP) for 1 hr, prior to antibody staining. The experiment was repeated three times and a representative blot is shown.

Article Snippet: Purified, anisomycin-activated JNK (1 μg, Signal Chem, M34-10G) was incubated with 5 μg bacterially purified recombinant SIRT6 for 2 hr in Kinase Reaction Buffers (Signal Chem, K23-09-05 and K21-09-05) and ATP or [γ- 32 P]-ATP.

Techniques: In Vitro, Phospho-proteomics, Purification, Incubation, Expressing, Plasmid Preparation, Mutagenesis, Inhibition, Western Blot, Stable Transfection, Construct, Activity Assay, In Vivo, Generated, Transfection, Staining

Journal: Cell reports

Article Title: JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

doi: 10.1016/j.celrep.2016.08.006

Figure Lengend Snippet: (A) Chromatin-enriched fractions from WT MEFs indicate that SIRT6 is rapidly recruited to chromatin following exposure of cells to paraquat. Cells were treated with 0.5 mM paraquat, and chromatin-enriched extracts were prepared at the indicated time points (n = 3). A representative blot is shown. (B) Recruitment of SIRT6-GFP to sites of laser-induced DNA damage was monitored in U2OS cells transfected with SIRT6-GFP in the presence or absence of a JNK inhibitor (SP600125). Cells pretreated with 20 µM JNK inhibitor for 2 hr exhibited severe defects in their ability to recruit SIRT6 to DSB sites. Representative images are shown (n > 8 for each sample). Error bars indicate SEM. See also . (C) Recruitment of WT, S10A, or S10E SIRT6-GFP to sites of laser-induced DNA damage was monitored in U2OS cells. S10E SIRT6-GFP exhibited enhanced recruitment efficiency to sites of DSBs, whereas S10A SIRT6-GFP exhibited diminished recruitment efficiency to DSB sites. Representative images are shown (n > 8 for each sample). Error bars indicate SEM.

Article Snippet: Purified, anisomycin-activated JNK (1 μg, Signal Chem, M34-10G) was incubated with 5 μg bacterially purified recombinant SIRT6 for 2 hr in Kinase Reaction Buffers (Signal Chem, K23-09-05 and K21-09-05) and ATP or [γ- 32 P]-ATP.

Techniques: Transfection

Journal: Cell reports

Article Title: JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

doi: 10.1016/j.celrep.2016.08.006

Figure Lengend Snippet: (A) In vitro mono-ADP ribosylation reaction. Bacterially purified recombinant WT and S10A and S10E SIRT6 proteins were incubated with catalytically inactive recombinant PARP1 (C-terminal truncation, containing only aa 1–655) for 2 hr. S10E SIRT6 was able to more robustly mono-ADP ribosylate the PARP1 substrate than either WT or S10A SIRT6 (n = 3). A representative reaction is shown. (B) In vitro ribosylation assay demonstrating S10E SIRT6 more robustly stimulates PARP1 activity. Bacterially purified WT, S10A, or S10E SIRT6 was incubated with PARP1. PARP1 activity was measured by quantifying the amount of auto-poly-ADP ribosylation of the protein by immunoblotting with antibodies targeting poly-ADP ribose. The experiment was independently repeated three times; the right panel shows quantification; error bars indicate SD (*p < 0.05 and **p < 0.01).

Article Snippet: Purified, anisomycin-activated JNK (1 μg, Signal Chem, M34-10G) was incubated with 5 μg bacterially purified recombinant SIRT6 for 2 hr in Kinase Reaction Buffers (Signal Chem, K23-09-05 and K21-09-05) and ATP or [γ- 32 P]-ATP.

Techniques: In Vitro, Purification, Recombinant, Incubation, Activity Assay, Western Blot

Journal: Cell reports

Article Title: JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

doi: 10.1016/j.celrep.2016.08.006

Figure Lengend Snippet: (A) Recruitment of PARP1-GFP to sites of laser-induced DNA damage was measured in WT and SIRT6 KO MEFs. In the absence of SIRT6, PARP1 exhibited a striking failure to fully recruit to DNA break sites. Representative images are shown (n > 8 for each condition); error bars indicate SEM. (B) Recruitment of SIRT6-GFP to sites of laser-induced DNA damage was measured in WT and PARP1 KO MEFs. In the absence of PARP1, SIRT6 was able to be fully recruited to DNA break sites. Representative images are shown (n > 8 for each condition). Error bars represent SEM (n.s., not significant). (C) Recruitment of PARP1-GFP to sites of laser-induced DNA damage in MEFs overexpressing WT, S10A, or S10E SIRT6. Cells overexpressing WT SIRT6 and S10E SIRT6 were able to more robustly recruit PARP1-GFP to sites of DNA damage. By contrast, cells overexpressing S10A SIRT6 failed to stimulate PARP1 recruitment to DNA damage sites. Error bars indicate SEM (n > 8). (D) Modification of K521A PARP1 is required for the efficient recruitment of PARP1 to DSB sites. Recruitment of PARP-GFP1 or K521A PARP1-GFP to sites of laser-induced DNA damage was measured in MEFs. K521A PARP1-GFP exhibited a failure to efficiently recruit to DNA damage sites. Representative images are shown (n > 8 for each condition). Error bars indicate SEM (n > 8). (E) JNK inhibition abrogates PARP1 recruitment to DNA damage sites. Recruitment of PARP1-GFP to sites of laser-induced DNA damage was measured in WT MEFs in the presence or absence of a JNK inhibitor. JNK inhibition with SP600125 resulted in failure to recruit PARP1 to DNA damage sites. Error bars indicate SEM (n > 8).

Article Snippet: Purified, anisomycin-activated JNK (1 μg, Signal Chem, M34-10G) was incubated with 5 μg bacterially purified recombinant SIRT6 for 2 hr in Kinase Reaction Buffers (Signal Chem, K23-09-05 and K21-09-05) and ATP or [γ- 32 P]-ATP.

Techniques: Modification, Inhibition

Journal: Cell reports

Article Title: JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

doi: 10.1016/j.celrep.2016.08.006

Figure Lengend Snippet: Upon oxidative stress JNK phosphorylates SIRT6 on Serine 10. This results in rapid recruitment of SIRT6 to the DSB site and simultaneously stimulates SIRT6 mono-ADP ribosylation of PARP1. PARP1 mono-ADP ribosylation leads to recruitment of PARP1 to DSB site and activates PARP1 poly-ADP ribosylation activity. This sequence of events represents the initial steps in the assembly of repair machinery on a DSB, and it is required for efficient DSB repair under oxidative stress conditions.

Article Snippet: Purified, anisomycin-activated JNK (1 μg, Signal Chem, M34-10G) was incubated with 5 μg bacterially purified recombinant SIRT6 for 2 hr in Kinase Reaction Buffers (Signal Chem, K23-09-05 and K21-09-05) and ATP or [γ- 32 P]-ATP.

Techniques: Activity Assay, Sequencing

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: SIRT6 Ameliorates Cancer Cachexia–Associated Adipose Wasting by Suppressing TNFR2 Signalling in Mice

doi: 10.1002/jcsm.13734

Figure Lengend Snippet: Serum SIRT6 concentrations are associated with cachexia in patients with cancer. (A) Diagram elucidating the collection and analysis of patient serum sample. Serum collected from healthy controls, cachectic cancer patients and non‐cachectic cancer patients, were used to measure SIRT6 concentrations by enzyme‐linked immunosorbent assay (ELISA). (B) Serum SIRT6 concentrations of healthy controls ( n = 22) and cancer patients ( n = 22) were compared. (C) Serum SIRT6 concentrations of cachectic cancer patients ( n = 12) and non‐cachectic cancer patients ( n = 10) were compared. ** p < 0.01.

Article Snippet: Human serum SIRT6, TNFα and TNFR2 concentrations were analysed by Human NAD‐dependent deacetylase sirtuin‐6 (SIRT6) ELISA kit (CSB‐E17018h, CUSABIO), human tumour necrosis factor α (TNF‐α) ELISA kit (CSB‐E04740h, CUSABIO) and human soluble tumour necrosis factor receptor 2 (sTNF‐R2) ELISA kit (CSB‐E11266h, CUSABIO) according to the manufacturer's instructions, individually.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: SIRT6 Ameliorates Cancer Cachexia–Associated Adipose Wasting by Suppressing TNFR2 Signalling in Mice

doi: 10.1002/jcsm.13734

Figure Lengend Snippet: SIRT6 overexpression prevents body weight loss and adipose tissue wasting in tumour‐bearing mice. (A–D) SIRT6 transgenic (TG) and wild type (WT) mice were inoculated with LLC cells or PBS and euthanized 21 days after tumour injection. Tumour weight (A), carcass weight (B), weight of total fat tissue (C), weight of epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT) and brown adipose tissue (BAT) (D) were measured ( n = 6 per group). (E) Representative H & E‐stained images of eWAT, iWAT and BAT. Adipocytes size distribution in eWAT and iWAT and ratio of lipid area in BAT were quantified ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human serum SIRT6, TNFα and TNFR2 concentrations were analysed by Human NAD‐dependent deacetylase sirtuin‐6 (SIRT6) ELISA kit (CSB‐E17018h, CUSABIO), human tumour necrosis factor α (TNF‐α) ELISA kit (CSB‐E04740h, CUSABIO) and human soluble tumour necrosis factor receptor 2 (sTNF‐R2) ELISA kit (CSB‐E11266h, CUSABIO) according to the manufacturer's instructions, individually.

Techniques: Over Expression, Transgenic Assay, Injection, Staining

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: SIRT6 Ameliorates Cancer Cachexia–Associated Adipose Wasting by Suppressing TNFR2 Signalling in Mice

doi: 10.1002/jcsm.13734

Figure Lengend Snippet: SIRT6 overexpression decreased the expression of genes involved of browning and lipolysis. (A) The mRNA levels of UCP1 in the eWAT were determined by qRT‐PCR ( n = 4 per group). (B) The mRNA levels of PGC1α in the eWAT were determined by qRT‐PCR ( n = 4 per group). (C) The expression and phosphorylation levels of lipolysis associated proteins in the eWAT from WT + PBS, WT + LLC, TG + PBS and TG + LLC mice were determined by western blot ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human serum SIRT6, TNFα and TNFR2 concentrations were analysed by Human NAD‐dependent deacetylase sirtuin‐6 (SIRT6) ELISA kit (CSB‐E17018h, CUSABIO), human tumour necrosis factor α (TNF‐α) ELISA kit (CSB‐E04740h, CUSABIO) and human soluble tumour necrosis factor receptor 2 (sTNF‐R2) ELISA kit (CSB‐E11266h, CUSABIO) according to the manufacturer's instructions, individually.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Phospho-proteomics, Western Blot

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: SIRT6 Ameliorates Cancer Cachexia–Associated Adipose Wasting by Suppressing TNFR2 Signalling in Mice

doi: 10.1002/jcsm.13734

Figure Lengend Snippet: The effect of SIRT6 overexpression and knockout on adipocytes lipolysis. (A) Mouse embryonic fibroblasts (MEFs) isolated from SIRT6 transgenic (TG) and wild type (WT) embryos were induced differentiation into mature adipocytes. Adipocytes were treated for 24 h with LLC cell‐conditioned medium (LLC‐CM) or control medium (DMEM). Representative images of oil red O staining were shown. Mouse embryonic fibroblasts (MEFs) isolated from SIRT6 knockout (KO) and wild type (WT) embryos were induced differentiation into mature adipocytes. Adipocytes were treated for 24 h with LLC cell‐conditioned medium (LLC‐CM) or control medium (DMEM). Representative images of oil red O staining were shown. (B) The quantification of lipid content was shown ( n = 4 per group). (C) Relative glycerol release in medium supernatant were measured ( n = 4 per group). (D) The expression of ATGL and phosphorylation levels of HSL in different adipocytes were determined by western blot ( n = 4 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human serum SIRT6, TNFα and TNFR2 concentrations were analysed by Human NAD‐dependent deacetylase sirtuin‐6 (SIRT6) ELISA kit (CSB‐E17018h, CUSABIO), human tumour necrosis factor α (TNF‐α) ELISA kit (CSB‐E04740h, CUSABIO) and human soluble tumour necrosis factor receptor 2 (sTNF‐R2) ELISA kit (CSB‐E11266h, CUSABIO) according to the manufacturer's instructions, individually.

Techniques: Over Expression, Knock-Out, Isolation, Transgenic Assay, Control, Staining, Expressing, Phospho-proteomics, Western Blot

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: SIRT6 Ameliorates Cancer Cachexia–Associated Adipose Wasting by Suppressing TNFR2 Signalling in Mice

doi: 10.1002/jcsm.13734

Figure Lengend Snippet: SIRT6 functions in lipolysis mediated by TNFR2. (A–B) Tumour necrosis factor receptor (TNFR1 and TNFR2) protein levels in the eWAT from WT + PBS, WT + LLC, SIRT6 TG + PBS and SIRT6 TG + LLC mice were determined by western blot ( n = 6 per group). (C) Serum TNFR2 concentrations in mice were measured by ELISA (PBS, n = 7 per group; LLC, n = 11 per group). (D) cAMP levels in WT + DMEM, WT + LLC‐CM, TG + DMEM, TG + LLC‐CM adipocytes ( n = 3 per group) or WT + DMEM, WT + LLC‐CM, KO + DMEM, KO + LLC‐CM adipocytes ( n = 4 per group) were measured. WT + DMEM (WT adipocytes were treated with DMEM medium), WT + LLC‐CM (WT adipocytes were treated with LLC cell‐conditioned medium), TG + DMEM (SIRT6 TG adipocytes were treated with DMEM medium), TG + LLC‐CM (SIRT6 TG adipocytes were treated with LLC cell‐conditioned medium), KO + DMEM (SIRT6 KO adipocytes were treated with DMEM medium) and KO + LLC‐CM (SIRT6 KO adipocytes were treated with LLC cell‐conditioned medium). (E) MEFs isolated from KO and WT embryos were induced differentiation into mature adipocytes. Adipocytes were treated for 24 h with DMEM, LLC cell‐conditioned medium and a combination of LLC cell‐conditioned medium and 12.5 μg/mL TNFR2 antagonist. Representative images of oil red O staining and the quantification of lipid content were shown ( n = 4 per group). (F) Serum TNFα and TNFR2 concentrations of cachectic cancer patients ( n = 12) and non‐cachectic cancer patients ( n = 10) were compared. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human serum SIRT6, TNFα and TNFR2 concentrations were analysed by Human NAD‐dependent deacetylase sirtuin‐6 (SIRT6) ELISA kit (CSB‐E17018h, CUSABIO), human tumour necrosis factor α (TNF‐α) ELISA kit (CSB‐E04740h, CUSABIO) and human soluble tumour necrosis factor receptor 2 (sTNF‐R2) ELISA kit (CSB‐E11266h, CUSABIO) according to the manufacturer's instructions, individually.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, Staining

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: SIRT6 Ameliorates Cancer Cachexia–Associated Adipose Wasting by Suppressing TNFR2 Signalling in Mice

doi: 10.1002/jcsm.13734

Figure Lengend Snippet: MDL800 reverses LLC‐induced adipocytes lipolysis. (A) Western blot analysis of SIRT6 and its substrates H3K9ac and H3K56ac in WT adipocytes with or without 20‐μM MDL‐800 treatment for 24 h ( n = 3 per group). (B) MEFs isolated from WT embryos were induced differentiation into mature adipocytes. Adipocytes were treated for 24 h with DMEM, LLC cell‐conditioned medium and a combination of LLC cell‐conditioned medium and 20‐μM MDL800. Representative images of oil red O staining and the quantification of lipid content were shown ( n = 3 per group). (C) Glycerol levels in medium supernatant were measured ( n = 6 per group). (D) The expression and phosphorylation levels of lipolysis associated proteins in the WT + DMEM, WT + LLC‐CM and WT + LLC‐CM + 20‐μM MDL800 were determined by western blot ( n = 4–6 per group). (E) cAMP levels in WT + DMEM, WT + LLC‐CM and WT + LLC‐CM + 20 μM MDL800 adipocytes were measured ( n = 3 per group). (F) Role of SIRT6 in cancer cachexia. TNFα, secreted from tumour cell, interacts with membrane TNFR2 and then induces the activation of lipolysis signalling pathway, mainly indicated by increased expression of ATGL and over‐phosphorylation of HSL and Perilipin 1. Without SIRT6, lipolysis is enhanced due to increased expression of TNFR2 and interaction of TNFα and TNFR2, which led to adipose wasting in tumour‐bearing mice. When SIRT6 is overexpressed or pharmaceutically activated, decreased expression of TNFR2 attenuates the activation of lipolysis signalling, which led to adipose homeostasis and then metabolic balance in mice. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human serum SIRT6, TNFα and TNFR2 concentrations were analysed by Human NAD‐dependent deacetylase sirtuin‐6 (SIRT6) ELISA kit (CSB‐E17018h, CUSABIO), human tumour necrosis factor α (TNF‐α) ELISA kit (CSB‐E04740h, CUSABIO) and human soluble tumour necrosis factor receptor 2 (sTNF‐R2) ELISA kit (CSB‐E11266h, CUSABIO) according to the manufacturer's instructions, individually.

Techniques: Western Blot, Isolation, Staining, Expressing, Phospho-proteomics, Membrane, Activation Assay