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: Figure 2. 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 trans- fected 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, Control, 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: Figure 4. 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 mM) or the autophagy agonist rapamycin (Rapa, 5 mM), 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 treat- ments 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 show- ing 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, Control, 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: Figure 5. SIRT6 regulated autophagy in melanoma. (A) Immunoblotting analysis showing the expression of autophagy markers LC3 and SQSTM1 in melanoma cells trans- fected with overexpression vectors (SIRT6-WTs), mutant vectors (SIRT6H133Y) and shRNA vectors followed by treatment with the autophagosome degradation inhibitor chloroquine (CQ, 10 mM) 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; F, P <0.05, SIR- T6H133Y 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 mm (upper row of each cell line) and 60 mm (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; F, 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, Over Expression, Mutagenesis, shRNA, Control, Transfection, 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: Figure 7. 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 mM). (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 inhib- itor (0.5 mM). (D) Apoptosis analysis by flow cytometry in melanoma cells with different treatments as described in (B). Statistical charts represent 3 individual experi- ments. 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, Knockdown, Plasmid Preparation, Transfection, CCK-8 Assay, shRNA, Control, Inhibition, 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: Figure 8. The bimodal role of SIRT6 in melanoma growth at different stages in vivo. (A) Primary and metastatic melanoma cells stably transfected with SIRT6 overexpres- sion 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 for- mula (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 representa- tively (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, shRNA, Injection, Control, Western Blot, Plasmid Preparation

Journal: Osteoarthritis and cartilage

Article Title: Depletion of SIRT6 causes cellular senescence, DNA damage, and telomere dysfunction in human chondrocytes.

doi: 10.1016/j.joca.2015.03.024

Figure Lengend Snippet: Fig. 1. SIRT6 expression in articular cartilages and cultured chondrocytes (A) Microscopic images of Safranin-O, Immunohistochemistry for SIRT6 and negative control (scale bars ¼ 100 mm). Cartilage samples are MFC and LFC from a 72-year-old female patient with varus OA, and femoral head (FH) from a 74-year-old female patient with femoral neck fracture. The chondrocytes with brown-colored nucleus were positive. Representative results were obtained from six different patients for FH and six different OA patients for MFC and LFC. (B) A microscopic image of immunohistochemistry of the FH for SIRT6 taken at a high magnification (400). SIRT6 was localized to the nucleus (Scale bar ¼ 100 mm). (C) Microscopic images of cultured human chondrocytes immunostained with antibodies against SIRT6 (red) counterstained with DAPI (blue), and merged images of DAPI and SIRT6 (scale bars ¼ 20 mm). Immunofluorescence analysis showed that SIRT6 was expressed in human chondrocytes and localized to the nucleus. Representative results were obtained from repeated experiment using human chondrocytes from three different donors.

Article Snippet: The following antibodies were used in this study: rabbit antihuman SIRT6 antibody, rabbit anti-human p21 antibody (both from Cell Signaling Technology), and mouse anti-human p16 antibody (Santa Cruz); and HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG (both from GE Healthcare).

Techniques: Expressing, Cell Culture, Immunohistochemistry, Negative Control

Journal: Osteoarthritis and cartilage

Article Title: Depletion of SIRT6 causes cellular senescence, DNA damage, and telomere dysfunction in human chondrocytes.

doi: 10.1016/j.joca.2015.03.024

Figure Lengend Snippet: Fig. 2. The efficiency and influence of SIRT6 inhibition by SIRT6 siRNA (A) Real-time PCR analysis for SIRT6 mRNA in normal human chondrocytes. The SIRT6 mRNA level was significantly reduced to approximately 40% by SIRT6 siRNA transfection. Non-silencing siRNA was used as a control. *Statistically significant difference (n ¼ 3, P ¼ 0.001). GAPDH was used as endogenous control. The value for control expression level/GAPDH was set as 1. Values are the mean ± 95% CI. (B) Western blotting analysis for SIRT6 in normal human chondrocytes. Tubulin was served as a control. Representative results are shown from three different donors. (C) Real-time PCR analysis for the expression of MMP family members. MMP-1 and 13 mRNA were significantly increased by the SIRT6 depletion. GAPDH was used as endogenous control. The value for control siRNA expression level/GAPDH was set as 1. Values are the mean ± 95% CI. *Statistically significant difference (n ¼ 3, P ¼ 0.03 and P ¼ 0.01 respectively).

Article Snippet: The following antibodies were used in this study: rabbit antihuman SIRT6 antibody, rabbit anti-human p21 antibody (both from Cell Signaling Technology), and mouse anti-human p16 antibody (Santa Cruz); and HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG (both from GE Healthcare).

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Transfection, Control, Expressing, Western Blot

Journal: Osteoarthritis and cartilage

Article Title: Depletion of SIRT6 causes cellular senescence, DNA damage, and telomere dysfunction in human chondrocytes.

doi: 10.1016/j.joca.2015.03.024

Figure Lengend Snippet: Fig. 3. The influence of the depletion of SIRT6 on proliferation and senescence. (A) The expression of SIRT6 and PCNA in articular cartilage of MFC from a 72-year-old female patient with varus OA. Safranin-O, Immunohistochemistry for SIRT6 at 40 and 100, and for PCNA at 100. SIRT6 was strongly expressed in chondrocytes forming clusters that also express PCNA. (Scale bars ¼ 100 mm). Representative results are shown from six repeated experiments from six different patients. (B) Proliferation activity assay using Cell Counting Kit-8. Depletion of SIRT6 significantly reduced proliferation activity 72 and 96 h after lipofection. Values are the mean ± 95% CI of the data obtained from three donors. *Statistically significant difference (n ¼ 3, P ¼ 0.02 and P ¼ 0.002 respectively). (C) SA-b-Gal assay. The percentage of SA-b-Gal positive cells was significantly higher in the SIRT6 siRNA group compared with the control group. Values are the mean ± 95% CI of the data obtained from three donors. (Scale bar ¼ 100 mm). *Statistically significant difference (n ¼ 3, P ¼ 0.008).

Article Snippet: The following antibodies were used in this study: rabbit antihuman SIRT6 antibody, rabbit anti-human p21 antibody (both from Cell Signaling Technology), and mouse anti-human p16 antibody (Santa Cruz); and HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG (both from GE Healthcare).

Techniques: Expressing, Immunohistochemistry, Activity Assay, Cell Counting, Control

Journal: Osteoarthritis and cartilage

Article Title: Depletion of SIRT6 causes cellular senescence, DNA damage, and telomere dysfunction in human chondrocytes.

doi: 10.1016/j.joca.2015.03.024

Figure Lengend Snippet: Fig. 4. The influence of the depletion of SIRT6 on DNA damage and telomere dysfunction. (A) Microscopic images of control siRNA and SIRT6 siRNA chondrocytes immunostained with antibodies against gH2AX (red) and TRF-1 (green), and counterstained with DAPI (blue). White arrows in the merged images point to sites of co-localization which means TIFs. Scale bars ¼ 5 mm gH2AX foci and TIFs were increased in the SIRT6-depleted chondrocytes than in controls. Representative results are shown from four repeated experiments using chondrocytes from three different donors. (B) Quantification of gH2AX foci expressed as mean relative area per cell. Twenty nuclei from the chondrocytes transfected with SIRT6 siRNA and control siRNA were examined. The relative area of gH2AX foci was significantly greater in the SIRT6-depleted chondrocytes than in control chondrocytes. Values are the mean ± 95% CI. *Statistically significant difference (n ¼ 3, P ¼ 0.0001). (C) Quantification of TIFs per cell. Twenty nuclei from the chondrocytes transfected with SIRT6 siRNA and control siRNA were examined in each donor. The average number of TIFs per cell was significantly higher in the SIRT6-depleted chondrocytes than in control chondrocytes. Values are the mean ± 95% CI. *Statistically significant difference (n ¼ 3, P ¼ 0.007).

Article Snippet: The following antibodies were used in this study: rabbit antihuman SIRT6 antibody, rabbit anti-human p21 antibody (both from Cell Signaling Technology), and mouse anti-human p16 antibody (Santa Cruz); and HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG (both from GE Healthcare).

Techniques: Control, Transfection

Journal: Osteoarthritis and cartilage

Article Title: Depletion of SIRT6 causes cellular senescence, DNA damage, and telomere dysfunction in human chondrocytes.

doi: 10.1016/j.joca.2015.03.024

Figure Lengend Snippet: Fig. 5. Western blotting for p16 and p21. The p16 protein level was higher and the p21 protein level was lower in the SIRT6-depleted chondrocytes than in control chon- drocytes. Representative results were obtained from three different donors. Tubulin was used as a control.

Article Snippet: The following antibodies were used in this study: rabbit antihuman SIRT6 antibody, rabbit anti-human p21 antibody (both from Cell Signaling Technology), and mouse anti-human p16 antibody (Santa Cruz); and HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG (both from GE Healthcare).

Techniques: Western Blot, Control

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: Figure 1. Aberrant SIRT6 expression in melanoma at different stages. (A) qRT-PCR analysis of SIRT6 mRNA levels in benign nevus tissues (left group, benign nevus, n = 12) and melanoma tissues at different stages (middle group, primary melanoma, n = 12; right group, metastatic melanoma, n = 16). BN, benign nevus; PM, primary mela- noma; MM, metastatic melanoma. , P < 0.001. (B) Representative immunoblotting analysis of SIRT6 expression in the 3 groups as described previously (n = 3 per group). The lower panel is a densitometry analysis of 3 individual experiments. Data are presented as means § SD. , P < 0.01. (C) Representative immunofluorescence staining images of SIRT6 expression and localization in the 3 groups as described previously. Scale bars: 60 mm, left panel; 20 mm, right panel. (D) qRT-PCR analysis of SIRT6 mRNA level in normal human melanocytes (MCs) and melanoma cell lines at different stages. The relative SIRT6 mRNA abundance in MCs was designated as 1 (n = 3). Data are presented as means § SD. , P < 0.01; , P < 0.001. (E) Immunoblotting analysis of SIRT6 expression in MCs and melanoma cell lines at different stages. The lower panel is a densitometry analysis of 3 individual experiments. Data are presented as means § SD. , P< 0.05; , P < 0.01. For (A, B, D and E), a 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: Expressing, Quantitative RT-PCR, Western Blot, 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: Figure 2. 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 trans- fected 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, Control, 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: Figure 3. Significant correlation between SIRT6 expression and autophagy level in melanoma. (A) Immunoblotting analysis of the expression of autophagy markers LC3 and SQSTM1 in benign nevus tissues, primary and metastatic melanoma tissues (n = 3 per group). The right panels are densitometry analysis of 3 individual experiments. Data are presented as means § SD. , P < 0.01, BN as control. (B) Immunoblotting analysis of the expression of autophagy markers in MCs and melanoma cell lines at dif- ferent stages. The right panels are densitometry analysis of 3 individual experiments. Data are presented as means § SD. , P< 0.05; , P < 0.01; , P < 0.001, MC as control. (C) Immunofluorescence staining images of LC3 through confocal microscopy analysis in benign nevus tissues and melanoma tissues at different stages. Scale bar: 60 mm. (D) Spearman’s correlation test of immunoblotting densitometry data between relative SIRT6 and LC3-II:LC3-I ratio expression in benign nevus tissues and melanoma tissues at different stages (n = 30, 10 per group). (E) Spearman’s correlation test of immunoblotting densitometry data between relative SIRT6 and SQSTM1 expression in the same samples as indicated in (D). 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: Expressing, Western Blot, Control, Staining, 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: Figure 4. 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 mM) or the autophagy agonist rapamycin (Rapa, 5 mM), 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 treat- ments 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 show- ing 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, Control, 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: Figure 5. SIRT6 regulated autophagy in melanoma. (A) Immunoblotting analysis showing the expression of autophagy markers LC3 and SQSTM1 in melanoma cells trans- fected with overexpression vectors (SIRT6-WTs), mutant vectors (SIRT6H133Y) and shRNA vectors followed by treatment with the autophagosome degradation inhibitor chloroquine (CQ, 10 mM) 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; F, P <0.05, SIR- T6H133Y 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 mm (upper row of each cell line) and 60 mm (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; F, 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, Over Expression, Mutagenesis, shRNA, Control, Transfection, 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: Figure 6. SIRT6 inhibited IGF-AKT signaling in melanoma. (A and B) qRT-PCR analysis of IGF1R mRNA level in melanoma tissues and cell lines. Data are presented as means § SD. , P < 0.05; , P < 0.01; , P < 0.001, BN and MC as control, respectively. (C and D) Immunoblotting analysis showing the expressions of IGF-AKT signaling-related proteins in melanoma tissues and cell lines at different stages. BN as control in (C), MCs as control in (D). The ‘p-’ prefix indicates the phosphorylated form. (E and F) Spear- man’s correlation test of immunoblotting densitometry analysis between SIRT6 and the ratio of p-IGF1R:IGF1R or p-AKT:AKT in melanoma tissues at different stages and benign nevus tissues. (n = 30, 10 per group.) (G and H) Representative immunoblotting analysis showing the expression of IGF-AKT signaling-related proteins in mela- noma cells with different treatments as indicated. 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: Quantitative RT-PCR, Control, Western Blot, Expressing

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: Figure 7. 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 mM). (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 inhib- itor (0.5 mM). (D) Apoptosis analysis by flow cytometry in melanoma cells with different treatments as described in (B). Statistical charts represent 3 individual experi- ments. 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, Knockdown, Plasmid Preparation, Transfection, CCK-8 Assay, shRNA, Control, Inhibition, 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: Figure 8. The bimodal role of SIRT6 in melanoma growth at different stages in vivo. (A) Primary and metastatic melanoma cells stably transfected with SIRT6 overexpres- sion 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 for- mula (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 representa- tively (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, shRNA, Injection, Control, Western Blot, Plasmid Preparation

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: Figure 9. Schematic representation of the effects of SIRT6-regulated autophagy on melanoma growth and the underlying mechanism. In benign nevus, melanocytes undergo limited proliferation with a physiological basal level of SIRT6 and autophagy. In the case of reduced SIRT6 expression, melanocytes can proliferate uncontrollably, migrate to the upper epidermis along the basement membrane, and subsequently penetrate to the dermis, which forms primary melanoma with a lower autophagy level. Later, melanoma cells can enter into blood vessels and develop distant metastases, with SIRT6 and downstream autophagy remarkably increased. The effect of SIRT6 on autophagy in melanoma is mediated by IGF-AKT signaling, with the deacetylase activity of SIRT6 indispensable for the regulation.

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

Techniques: Expressing, Membrane, Histone Deacetylase Assay, Activity Assay