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

Journal: Autophagy

Article Title: Aberrant SIRT6 expression contributes to melanoma growth: Role of the autophagy paradox and IGF-AKT signaling

doi: 10.1080/15548627.2017.1384886

Figure Lengend Snippet: The bimodal role of SIRT6 in melanoma growth. (A) Primary (WM35 and WM793B) and metastatic (A2058 and A375) melanoma cell lines were stably transfected with SIRT6 overexpression vectors or shRNA vectors as indicated. Cells were reseeded and cell viability at the indicated time points was then measured by CCK8 assay. Data at each time point are presented as means ± SD and then successively connected into a growth curve. **, P < 0.01, FLAG and Ctr-shRNA as control respectively; ns, no significant difference. SIRT6-WT1 and SIRT6-WT2, FLAG-tagged overexpression vectors encoding SIRT6; FLAG, FLAG-tagged empty vector; SIRT6-shRNA1 and SIRT6-shRNA2, shRNA vectors against SIRT6; Ctr-shRNA, empty shRNA vector. (B) Cell cycle distributions were analyzed at 48 h in primary and metastatic melanoma cells stably transfected with the indicated vectors. Statistical charts represent 3 individual experiments. Data are presented as means ± SD. FLAG and Ctr-shRNA are controls. (C) Flow cytometry analysis of apoptosis at 24 h by ANXA5/Annexin V (an indicator of apoptosis) and 7-AAD staining in primary and metastatic melanoma cells stably transfected with overexpression vectors or shRNA vectors as indicated. Statistical charts represent 3 individual experiments. Data are presented as means ± SD. *, P < 0.05, FLAG and Ctr-shRNA are controls; ns, no significant difference. For (A and C), Students' t test was used to calculate the P value.

Article Snippet: Lentiviral vectors synthesizing SIRT6 shRNA or control shRNA were purchased from Ori Gene (TL317201).

Techniques: Stable Transfection, Transfection, Over Expression, shRNA, CCK-8 Assay, Plasmid Preparation, Flow Cytometry, Staining

Journal: Autophagy

Article Title: Aberrant SIRT6 expression contributes to melanoma growth: Role of the autophagy paradox and IGF-AKT signaling

doi: 10.1080/15548627.2017.1384886

Figure Lengend Snippet: SIRT6 regulated melanoma growth in an autophagy-dependent manner. (A) Primary (WM35 and WM793B) and metastatic (A2058 and A375) melanoma cell lines were stably transfected with SIRT6 overexpression vectors or shRNA vectors as indicated. Cells were reseeded and after 24 h treated with the autophagosome degradation inhibitor chloroquine (CQ, 10 μM) or the autophagy agonist rapamycin (Rapa, 5 μM), respectively, for subsequent CCK8 assay at the indicated time points. Data at each time point are presented as means ± SD and then successively connected into a growth curve. **, P < 0.01, FLAG and Ctr-shRNA as control respectively; #, P < 0.05; ##, P < 0.01, SIRT6-WT1 and SIRT6-WT2, as well as SIRT6-shRNA1 and SIRT6-shRNA2 as corresponding control, respectively; ns, no significant difference. WT1 and WT2, SIRT6-WT1 and SIRT6-WT2; shRNA1 and shRNA2, SIRT6-shRNA1 and SIRT6-shRNA2. (B) Cell cycle distributions were analyzed in melanoma cells at 48 h with the same treatments as described in (A). Statistical charts represent 3 individual experiments. Data are presented as means ± SD. FLAG and Ctr-shRNA, as well as SIRT6-WTs and SIRT6-shRNAs were regarded as corresponding control respectively. (C) Flow cytometry analysis of apoptosis by ANXA5/Annexin V and 7-AAD staining in different melanoma cells at 24 h with the same treatments as described in (A). Statistical charts represent 3 individual experiments. Data are presented as means ± SD. *, P < 0.05, FLAG and Ctr-shRNA are contols; #, P < 0.05, SIRT6-WT1 and SIRT6-WT2, as well as SIRT6-shRNA1 and SIRT6-shRNA2 as corresponding control, respectively; ns, no significant difference. (D) Immunoblotting analysis showing the expression of the apoptotic markers in melanoma cells with the indicated treatments. ACTB was used as a loading control. SIRT6H133Y, mutated vectors of SIRT6 with the loss of HDAC activity. C-PARP1, cleaved PARP1. For (A and C), Students' t test was used to calculate the P value.

Article Snippet: Lentiviral vectors synthesizing SIRT6 shRNA or control shRNA were purchased from Ori Gene (TL317201).

Techniques: Stable Transfection, Transfection, Over Expression, shRNA, CCK-8 Assay, Flow Cytometry, Staining, Western Blot, Expressing, Activity Assay

Journal: Autophagy

Article Title: Aberrant SIRT6 expression contributes to melanoma growth: Role of the autophagy paradox and IGF-AKT signaling

doi: 10.1080/15548627.2017.1384886

Figure Lengend Snippet: SIRT6 regulated autophagy in melanoma. (A) Immunoblotting analysis showing the expression of autophagy markers LC3 and SQSTM1 in melanoma cells transfected with overexpression vectors (SIRT6-WTs), mutant vectors (SIRT6H133Y) and shRNA vectors followed by treatment with the autophagosome degradation inhibitor chloroquine (CQ, 10 μM) as indicated. The lower panels are densitometry analysis of 3 individual experiments. Data are presented as means ± SD. *, P < 0.05, **, P < 0.01, blank group as control; #, P < 0.05, SIRT6-WT1 and SIRT6-WT2, as well as SIRT6-shRNA1 and SIRT6-shRNA2 as corresponding control, respectively; Φ, P <0.05, SIRT6H133Y as control; ns, no significant difference. (B) Autophagy reporter mRFP-LC3 plasmids were transiently transfected into melanoma cells followed by the treatments as described in (A). Representative images of fluorescent LC3 puncta (red) were photographed using confocal microscopy. Scale bar: 20 μm (upper row of each cell line) and 60 μm (lower row of each cell line). Numbers of RFP-LC3 puncta per cell were analyzed in the lower panels. Data are presented as means ± SD. **, P < 0.01, FLAG and Ctr-shRNA as control, respectively; #, P < 0.05, SIRT6-WT1 and SIRT6-shRNA1 as control, respectively; Φ, P <0.05, CQ-treated group as control. WT, SIRT6-WT1; shRNA, SIRT6-shRNA1, Ctr, Ctr-shRNA. For (A and B), Students' t test was used to calculate the P value.

Article Snippet: Lentiviral vectors synthesizing SIRT6 shRNA or control shRNA were purchased from Ori Gene (TL317201).

Techniques: Western Blot, Expressing, Transfection, Over Expression, Mutagenesis, shRNA, Confocal Microscopy

Journal: Autophagy

Article Title: Aberrant SIRT6 expression contributes to melanoma growth: Role of the autophagy paradox and IGF-AKT signaling

doi: 10.1080/15548627.2017.1384886

Figure Lengend Snippet: IGF-AKT signaling is involved in the regulation of autophagy by SIRT6 in melanoma. (A) Immunoblotting analysis showing the expression of autophagy markers (LC3 and SQSTM1) and IGF-AKT signaling-related proteins in melanoma cells with SIRT6 overexpression or knockdown as well as the indicated AKT interventions. MYR-HA-AKT1 and MYR-HA-AKT2, MYR-HA-tagged overexpression vectors encoding AKT1; MYR-HA-Ctr, MYR-HA-tagged empty vector; AKTi, AKT inhibitor (0.5 μM). (B) SIRT6-overexpressing primary melanoma cells and SIRT6-silencing metastatic melanoma cells were transfected with AKT overexpression vectors or treated with AKT inhibitor, respectively. After 24 h, cells were reseeded for CCK8 assay. Data at each time point are presented as means ± SD and then successively connected into a growth curve. *, P < 0.05, MYR-HA-Ctr and Ctr-shRNA as control, respectively; #, P < 0.05, SIRT6-WT1, SIRT6-shRNA1 and SIRT6-shRNA2 as control; ns, no significant difference. WT1, SIRT6-WT1; shRNA1 and shRNA2, SIRT6-shRNA1 and SIRT6-shRNA2; AKT1 and AKT2, MYR-HA-AKT1 and MYR-HA-AKT2. (C) Cell cycle distributions were analyzed at 48 h in melanoma cells with different treatments as described in (B). Statistical charts represent 3 individual experiments. Data are presented as means ± SD. MYR-HA-Ctr and Ctr-shRNA, as well as SIRT6-WT1 and SIRT6-shRNA1/2 as control. S1 and S2, SIRT6-shRNA1 and SIRT6-shRNA2; Ctr, MYR-HA-Ctr and Ctr-shRNA respectively; AKTi, AKT inhibitor (0.5 μM). (D) Apoptosis analysis by flow cytometry in melanoma cells with different treatments as described in (B). Statistical charts represent 3 individual experiments. Data are presented as means ± SD. *, P < 0.05, MYR-HA-Ctr and Ctr-shRNA as control respectively; #, P < 0.05, SIRT6-WT1, SIRT6-shRNA1 and SIRT6-shRNA2 as control; ns, no significant difference. For (B and D), Students' t test was used to calculate the P value.

Article Snippet: Lentiviral vectors synthesizing SIRT6 shRNA or control shRNA were purchased from Ori Gene (TL317201).

Techniques: Western Blot, Expressing, Over Expression, Plasmid Preparation, Transfection, CCK-8 Assay, shRNA, Flow Cytometry

Journal: Autophagy

Article Title: Aberrant SIRT6 expression contributes to melanoma growth: Role of the autophagy paradox and IGF-AKT signaling

doi: 10.1080/15548627.2017.1384886

Figure Lengend Snippet: The bimodal role of SIRT6 in melanoma growth at different stages in vivo. (A) Primary and metastatic melanoma cells stably transfected with SIRT6 overexpression vectors or SIRT6 shRNA vectors were subcutaneously injected into NOD/SCID nude mice (n = 5 per group) for the generation of subcutaneous xenograft tumors. Tumor volumes including tumor length (L) and width (W) were measured using vernier calipers every 3 d from d 6 after injection and then calculated according to the formula (L × W2)/2. Data are presented as means ± SD (from 5 individual mice). **, P < 0.01, FLAG and Ctr-shRNA as control, respectively. (B) Tumor weights were analyzed at the terminal time point. At the end of 33 d, tumors were excised and weighed. The data are shown as means ± SD (from 5 mice per group). **, P < 0.01, FLAG and Ctr-shRNA as control, respectively. (C and D) Tumors from sacrificed mice were dissected 33 d after subcutaneous injection and are shown in the indicated row representatively (n = 4 per group). (E and F) Representative immunoblotting analysis showing the expressions of IGF-AKT signaling-related proteins and autophagy markers (LC3 and SQSTM1) in tumors from sacrificed mice as indicated (n = 3 per group). WT1 and WT2, SIRT6-WT1 and SIRT6-WT2; FLAG, FLAG-tagged control vector; shRNA1 and shRNA2, SIRT6-shRNA1 and SIRT6-shRNA2; Ctr-shRNA, control shRNA. N, FLAG or Ctr-shRNA; W and T, SIRT6-WT1 and SIRT6-WT2; S and H, SIRT6-shRNA1 and SIRT6-shRNA2.

Article Snippet: Lentiviral vectors synthesizing SIRT6 shRNA or control shRNA were purchased from Ori Gene (TL317201).

Techniques: In Vivo, Stable Transfection, Transfection, Over Expression, shRNA, Injection, Western Blot, Plasmid Preparation