Journal: The Kaohsiung journal of medical sciences

Article Title: MiR-370 accelerated cerebral ischemia reperfusion injury via targeting SIRT6 and regulating Nrf2/ARE signal pathway.

doi: 10.1002/kjm2.12219

Figure Lengend Snippet: FIGURE 4 MiroRNA-370 (MiR-370) regulated Nrf2/ARE signal pathway via silencing information regulatory protein 6 (SIRT6). A, The mRNA level of SIRT6 was detected by qRT-PCR. B, The expression levels of SIRT6, Nrf2, HO-1, and NQO1 were determined by western blot. **P < .01

Article Snippet: The membrane was incubated with 5% skim milk for 1 hour and then immunoblotted with primary antibodies against SIRT6 (1:500; sc-517 196, Santa Cruz Biotechnology [SCBT]), β-actin (1:500; sc-47 778, SCBT, Dallas, Texas ), Nrf2 (1:500; sc-365 949, SCBT), HO-1 (1:400; ab69545, Abcam, Eugene, Oregon), NQO1 (1:400; ab28947, Abcam), Cleaved caspase-3 (1:500; sc-7272, SCBT) or Cleaved PARP (1:500; sc-56 196, SCBT) overnight at 4 C. Then, the membrane was washed with Tris-buffered saline with Tween 20 (TBST) for three times and incubated with m-IgGκ BP-HRP (mouse IgGκ binding protein-HRP; 1:5000; sc-8017, SCBT) at room temperature for 1 hour.

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: The Kaohsiung journal of medical sciences

Article Title: MiR-370 accelerated cerebral ischemia reperfusion injury via targeting SIRT6 and regulating Nrf2/ARE signal pathway.

doi: 10.1002/kjm2.12219

Figure Lengend Snippet: FIGURE 3 Silencing information regulatory protein 6 (SIRT6) was a direct target of microRNA (miR-370). A, The binding site of miR-370 and 30-UTR of SIRT6 mRNA. B, The transfection efficiency of miR-370 over-expression in HEK293 cells. C, The interaction of miR-370 and 30-UTR of SIRT6 mRNA was determined by luciferase activity assay in HEK293 cells. D, The mRNA level of SIRT6 was detected in SH-SY5Y cells by qRT- PCR. E, The protein level of SIRT6 was detected in SH-SY5Y cells by western blot. **P < .01

Article Snippet: The membrane was incubated with 5% skim milk for 1 hour and then immunoblotted with primary antibodies against SIRT6 (1:500; sc-517 196, Santa Cruz Biotechnology [SCBT]), β-actin (1:500; sc-47 778, SCBT, Dallas, Texas ), Nrf2 (1:500; sc-365 949, SCBT), HO-1 (1:400; ab69545, Abcam, Eugene, Oregon), NQO1 (1:400; ab28947, Abcam), Cleaved caspase-3 (1:500; sc-7272, SCBT) or Cleaved PARP (1:500; sc-56 196, SCBT) overnight at 4 C. Then, the membrane was washed with Tris-buffered saline with Tween 20 (TBST) for three times and incubated with m-IgGκ BP-HRP (mouse IgGκ binding protein-HRP; 1:5000; sc-8017, SCBT) at room temperature for 1 hour.

Techniques: Binding Assay, Transfection, Over Expression, Luciferase, Activity Assay, Quantitative RT-PCR, Western Blot

Journal: The Kaohsiung journal of medical sciences

Article Title: MiR-370 accelerated cerebral ischemia reperfusion injury via targeting SIRT6 and regulating Nrf2/ARE signal pathway.

doi: 10.1002/kjm2.12219

Figure Lengend Snippet: FIGURE 5 Overexpression of silencing information regulatory protein 6 (SIRT6) partially reversed the effect of miR-370 on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced SH-SY5Y cell injury. A, The cell viability was detected by CCK-8 assay in SH-SY5Y cells. B, The activity of lactate dehydrogenase (LDH) and the concentration of malondialdehyde (MDA) in SH-SY5Y cells were measured by commercial kit. C, The expression of Cleaved caspase-3 and Cleaved PARP were determined by western blot. **P < .01

Article Snippet: The membrane was incubated with 5% skim milk for 1 hour and then immunoblotted with primary antibodies against SIRT6 (1:500; sc-517 196, Santa Cruz Biotechnology [SCBT]), β-actin (1:500; sc-47 778, SCBT, Dallas, Texas ), Nrf2 (1:500; sc-365 949, SCBT), HO-1 (1:400; ab69545, Abcam, Eugene, Oregon), NQO1 (1:400; ab28947, Abcam), Cleaved caspase-3 (1:500; sc-7272, SCBT) or Cleaved PARP (1:500; sc-56 196, SCBT) overnight at 4 C. Then, the membrane was washed with Tris-buffered saline with Tween 20 (TBST) for three times and incubated with m-IgGκ BP-HRP (mouse IgGκ binding protein-HRP; 1:5000; sc-8017, SCBT) at room temperature for 1 hour.

Techniques: Over Expression, CCK-8 Assay, Activity Assay, Concentration Assay, Expressing, Western Blot

Journal: Scientific Reports

Article Title: ROS-Mediated Apoptotic Cell Death of Human Colon Cancer LoVo Cells by Milk δ-Valerobetaine

doi: 10.1038/s41598-020-65865-6

Figure Lengend Snippet: Upregulation of SIRT6 protein expression. ( a,b ) SIRT6 protein expression levels expressed as arbitrary units (AU) with * P < 0.05 vs Ctr, † P < 0.05 vs milk. Lane 1 = Ctr, lane 2 = milk, lane 3 = δVB, lane 4 = milk + δVB. ( c,d ) Representative confocal images of SIRT6 expression (red) and α-actin (green) in control cells (Ctr) and cells exposed to milk+ δVB for 72 h. Nuclei were counterstained with DAPI (blue). Scale Bar, insert = 200 μm; Enlarged = 50 μm.

Article Snippet: Antibody anti-p53 (orb323871), anti-cleaved-caspase 3 (orb106556) were bought from Biorbyt; Autophagy assay kit (ab139484), Rapamycin (ab139484); anti-SIRT6 (ab191385) and anti-β-actin (ab8227), anti-alpha tubulin (ab18251), Phalloidin-iFluor 488 Reagent (ab176753) were from Abcam (Cambridge CB2 0AX UK); anti-Bax (5023), anti-Bcl-2 (15071), anti-p21 (2947), anti-beclin-1 (4122), anti-cyclin A2 (4656), anti-caspase 3 (9662), anti-LC3B (2775), anti-p62 (SQSTM1) (5114), anti-cyclin B1 (4138), anti-caspase 9 (9508), anti-poly(ADP ribose) polymerase (PARP) (9532), anti-Atg7 (8558), anti- Caspase 8 (1C12) (9746); anti-α-tubulin (3873), anti-β-actin (3700) from Cell Signaling Technology (3 Trask Lane Danvers, MA, 01923 USA).

Techniques: Expressing

Journal: Scientific Reports

Article Title: ROS-Mediated Apoptotic Cell Death of Human Colon Cancer LoVo Cells by Milk δ-Valerobetaine

doi: 10.1038/s41598-020-65865-6

Figure Lengend Snippet: Apoptotic mechanism. ( a,b ) Percentage of apoptosis and representative dot plots of annexin V-FITC and PI-stained cells analyzed by flow cytometry. Data are expressed as mean ± SD of n = 4 experiments. At least 10.000 events were acquired. ( c – h ) Protein expression levels of caspase 3, caspase 9, PARP, Bax and Bcl-2 from LoVo cells treated for 72 h with milk (40% v/v), δVB (2 mM), milk+δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Lane 1 = Ctr, lane 2 = milk, lane 3 = δVB, lane 4 = milk + δVB. Analysis of densitometric intensity was calculated with Image J 1.52n version software. Arbitrary units of protein expression (AU) were quantified using α-tubulin or β-actin. Antibodies against Bax, Bcl-2 and SIRT6 (reported in Fig. ) were blotted on the same filter and quantified by using the same loading control (α-tubulin). ( i,j ) Flow cytometric analysis and representative dot plots of annexin V-FITC and PI double staining LoVo cells treated with caspase 9 inhibitor Z-LEHD-FMK (40 μM) or chloroquine (50 μM). Data are expressed as mean ± SD of n = 3 experiments. At least 10.000 events were acquired. ( k ) Cleaved caspase 3 protein expression level from LoVo cells treated for 72 h with milk+δVB, Z-LEHD-FMK + milk+δVB or HBSS-10 mM Hepes (40% v/v) (Ctr). Lane 1 = Ctr, lane 2 = milk + δVB, lane 3 = Z-LEHD-FMK + milk+δVB. ( l ) Protein expression levels of caspase 8 in LoVo cells after 72 h of treatment with milk (40% v/v), δVB (2 mM), milk+δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Lane 1=Ctr, lane 2 = milk, lane 3 = δVB, lane 4 = milk + δVB. * P < 0.05 vs Ctr, ** P < 0.01 vs Ctr, † P < 0.05 vs milk, †† P < 0.01 vs milk, + P < 0.05 vs milk+δVB, ++ P < 0.01 vs milk+δVB. The full-length blots are included in the supplementary information (Fig. ).

Article Snippet: Antibody anti-p53 (orb323871), anti-cleaved-caspase 3 (orb106556) were bought from Biorbyt; Autophagy assay kit (ab139484), Rapamycin (ab139484); anti-SIRT6 (ab191385) and anti-β-actin (ab8227), anti-alpha tubulin (ab18251), Phalloidin-iFluor 488 Reagent (ab176753) were from Abcam (Cambridge CB2 0AX UK); anti-Bax (5023), anti-Bcl-2 (15071), anti-p21 (2947), anti-beclin-1 (4122), anti-cyclin A2 (4656), anti-caspase 3 (9662), anti-LC3B (2775), anti-p62 (SQSTM1) (5114), anti-cyclin B1 (4138), anti-caspase 9 (9508), anti-poly(ADP ribose) polymerase (PARP) (9532), anti-Atg7 (8558), anti- Caspase 8 (1C12) (9746); anti-α-tubulin (3873), anti-β-actin (3700) from Cell Signaling Technology (3 Trask Lane Danvers, MA, 01923 USA).

Techniques: Staining, Flow Cytometry, Expressing, Software, Double Staining

Journal: Scientific Reports

Article Title: ROS-Mediated Apoptotic Cell Death of Human Colon Cancer LoVo Cells by Milk δ-Valerobetaine

doi: 10.1038/s41598-020-65865-6

Figure Lengend Snippet: Suppression of ROS production. ( a,b ) Flow cytometry analysis of green detection reagent staining following incubation with δVB (2 mM) and δVB + NAC. ** P < 0.01 vs Ctr, °P < 0.01 v s δVB. ( c, d ) Arbitrary units (AU) of SIRT6 protein expression in cells treated with δVB and milk+δVB in the presence of NAC. Lane 1 = Ctr, lane 2 = NAC + δVB, lane 3 = NAC + milk + δVB, * P < 0.05 vs Ctr. ( e ) SIRT6 protein expression in c e lls treated with RNAifectin transfection reagent (Vehicle), scramble siRNA (50 nM) (Scramble), SIRT6-siRNA (50 nM) or medium only (Ctr). Lane 1 = Ctr, lane 2 = Vehicle, lane 3 = Scramble, lane 4 = SIRT6-siRNA. ( f,g ) Flow cytometric autophagic activity performed by green detection reagent in LoVo cells treated for 72 h with milk+δVB, SIRT6-siRNA+milk+ δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Rapamycin (1 μM) was used as positive control. FlowJo V10 software was used to calculate median fluorescence intensities. ( h) Protein expression levels of LC3BI/ LC3BII from LoVo cells treated for 72 h with milk + δVB, SIRT6-siRNA + milk + δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Lane 1 = Ctr, lane 2 = milk + δVB, lane 3 = SIRT6-siRNA + milk + δVB. ( i,j ) Percentage of apoptosis and representative dot plots of annexin V-FITC and PI-stained cells analyzed by flow cytometry in LoVo cells after 72 h of treatment with milk+δVB, SIRT6-siRNA+milk+δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Data are expressed as mean ± SD of n = 4 experiments. At least 10.000 events were acquired. ( k ) Protein expression levels of PARP from LoVo cells treated for 72 h with milk + δVB, SIRT6-siRNA + milk + δVB, or HBSS-10 mM Hepes (40% v/v) (Ctr). Lane 1 = Ctr, lane 2 = milk + δVB, Lane 3 = SIRT6 siRNA + milk + δVB. * P < 0.05 vs Ctr, ** P < 0.01 vs Ctr, + P < 0.05 vs milk + δVB, ++ P < 0.01 vs milk + δVB.

Article Snippet: Antibody anti-p53 (orb323871), anti-cleaved-caspase 3 (orb106556) were bought from Biorbyt; Autophagy assay kit (ab139484), Rapamycin (ab139484); anti-SIRT6 (ab191385) and anti-β-actin (ab8227), anti-alpha tubulin (ab18251), Phalloidin-iFluor 488 Reagent (ab176753) were from Abcam (Cambridge CB2 0AX UK); anti-Bax (5023), anti-Bcl-2 (15071), anti-p21 (2947), anti-beclin-1 (4122), anti-cyclin A2 (4656), anti-caspase 3 (9662), anti-LC3B (2775), anti-p62 (SQSTM1) (5114), anti-cyclin B1 (4138), anti-caspase 9 (9508), anti-poly(ADP ribose) polymerase (PARP) (9532), anti-Atg7 (8558), anti- Caspase 8 (1C12) (9746); anti-α-tubulin (3873), anti-β-actin (3700) from Cell Signaling Technology (3 Trask Lane Danvers, MA, 01923 USA).

Techniques: Flow Cytometry, Staining, Incubation, Expressing, Transfection, Activity Assay, Positive Control, Software, Fluorescence

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: Custom rabbit polyclonal anti-SIRT6-phosphoS10 (pS10) antibodies were obtained from GenScript.

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: Custom rabbit polyclonal anti-SIRT6-phosphoS10 (pS10) antibodies were obtained from GenScript.

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: Custom rabbit polyclonal anti-SIRT6-phosphoS10 (pS10) antibodies were obtained from GenScript.

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: Custom rabbit polyclonal anti-SIRT6-phosphoS10 (pS10) antibodies were obtained from GenScript.

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: Custom rabbit polyclonal anti-SIRT6-phosphoS10 (pS10) antibodies were obtained from GenScript.

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: Custom rabbit polyclonal anti-SIRT6-phosphoS10 (pS10) antibodies were obtained from GenScript.

Techniques: Activity Assay, Sequencing

Journal: PloS one

Article Title: Identification of novel interacting partners of Sirtuin6.

doi: 10.1371/journal.pone.0051555

Figure Lengend Snippet: Figure 1. HEK293 cells were transiently transfected with Flag- tagged SIRT6 and analysed by immunocytochemistry and confocal microscopy. Panel A shows anti-Flag staining (red) and panel B the same cells counterstained with Hoechst33342 (blue). Panel C shows cells double stained with anti-RelA/p65 (green) and anti-Flag (red) and panel D only shows the corresponding anti-RelA/p65 staining alone. Transfected and untransfected cells showed similar gross morphology and clear nuclear localization of Flag-SIRT6. Scale bar = 10 mm. doi:10.1371/journal.pone.0051555.g001

Article Snippet: For MYBBP1A, RelA/p65, PIAS1 and SIRT6 immunoprecipitates SIRT6 total cell lysate was incubated with following antibodies bound to protein G Agarose overnight at 4uC: polyclonal rabbit anti MYBBP1A (Bethyl Lab A301-328A, lot 301-328A-1), rabbit anti RelA/p65 (Bethyl Lab A301-824A), anti PIAS1 (Abcam ab32219), anti SIRT6 (Bethyl LabA302-451A).

Techniques: Transfection, Immunocytochemistry, Confocal Microscopy, Staining

Journal: PloS one

Article Title: Identification of novel interacting partners of Sirtuin6.

doi: 10.1371/journal.pone.0051555

Figure Lengend Snippet: Figure 2. Interaction of SIRT6 with RelA/p65. HEK293 cells were transiently transfected with Flag-tagged SIRT6. Cell extracts were prepared and immunoprecipitated with an antibody to RelA/p65 and analysed by Western blotting (WB) with antibodies to RelA/p65 (lanes 1–4) SIRT6 (lane 5–8) and Flag (lane 9–12). Lanes 1,5 and 9 show the anti-RelA/p65 immunoprecipitate (p65 IP), lanes 2,6 and 10 show the non-immunoprecipitated or ‘‘flow through’’ material (FT), lanes 3,7 and 11 show a control immunoprecipitation using only Protein G agarose (p65 control) and lanes 4,8 and 12 are a sample of the total cell extract (input). doi:10.1371/journal.pone.0051555.g002

Article Snippet: For MYBBP1A, RelA/p65, PIAS1 and SIRT6 immunoprecipitates SIRT6 total cell lysate was incubated with following antibodies bound to protein G Agarose overnight at 4uC: polyclonal rabbit anti MYBBP1A (Bethyl Lab A301-328A, lot 301-328A-1), rabbit anti RelA/p65 (Bethyl Lab A301-824A), anti PIAS1 (Abcam ab32219), anti SIRT6 (Bethyl LabA302-451A).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control

Journal: PloS one

Article Title: Identification of novel interacting partners of Sirtuin6.

doi: 10.1371/journal.pone.0051555

Figure Lengend Snippet: Figure 3. HEK293 cells were transiently transfected with Flag- tagged wild type SIRT6 (lanes 1–4) or the Flag-tagged H133W mutant (lanes 6–9). Cell extracts were prepared and immunoprecip- itated with an anti-Flag antibody and analysed by Western blotting with anti-RelA/p65 antibody. Lanes 1 and 6 represent the total cell extracts used in the immunoprecipitation (input). Lanes 2 and 7 represent the anti-Flag immunoprecipitation eluted from the beads with Flag peptide (a-Flag IP). Lanes 3 and 8 represent the non-immunoprecipitated ‘‘flow through’’ material from the cell extract (FT) and lanes 4 and 9 represent the immunoprecipitated material eluted from the beads with SDS-PAGE sample buffer (a-Flag IP). Lane 5 contains molecular mass markers. doi:10.1371/journal.pone.0051555.g003

Article Snippet: For MYBBP1A, RelA/p65, PIAS1 and SIRT6 immunoprecipitates SIRT6 total cell lysate was incubated with following antibodies bound to protein G Agarose overnight at 4uC: polyclonal rabbit anti MYBBP1A (Bethyl Lab A301-328A, lot 301-328A-1), rabbit anti RelA/p65 (Bethyl Lab A301-824A), anti PIAS1 (Abcam ab32219), anti SIRT6 (Bethyl LabA302-451A).

Techniques: Transfection, Mutagenesis, Western Blot, Immunoprecipitation, SDS Page

Journal: PloS one

Article Title: Identification of novel interacting partners of Sirtuin6.

doi: 10.1371/journal.pone.0051555

Figure Lengend Snippet: Figure 4. Yeast-two-hybrid analysis. The coding sequence for full length wild type SIRT6 and the H133W mutant was PCR-amplified and cloned in frame with the LexA DNA binding domain (DBD) into the bait plasmid pB27. Fragments corresponding to amino acids 303–507 of PIAS1, amino acids 101–410 of TDG and amino acids 166–287 of TSPYL2 were extracted from the ULTImate Y2HTM human leukocyte and activated mononuclear cell library and cloned into the pB6 prey plasmid. Interactions were tested in growth assays as two independent clones (A and B) picked from each co- transformation except for the HGX positive control (columns 1 and 13, block A) and the empty controls (empty bait pB27 vector and empty prey vector pB6 columns 1 and 13, block B) that were only tested as a single clone. For each interaction several dilutions (1021, 1022, 1023 and 1024) of the diploid yeast culture normalized at 56104 cells and expressing both bait and prey constructs were spotted on the selective media. The left hand plate shows growth on media lacking tryptophan and leucine as a growth control test and to verify co-transformation of both plasmids. The same dilutions were spotted onto medium lacking tryptophan, leucine and histidine to confirm interaction of the bait and prey (right hand plate). For each interaction there is an empty bait (C-pB27) control co-transformation (columns 4, 7 and 10 for left hand plate and columns 16, 19 and 22 for the right hand plate). Column 5, 8, 11, 17, 20 and 23 have the H133W mutants cloned into the C-pB27 bait plasmid and columns 6, 9, 12, 18, 21 and 24 have the wild type SIRT6 cloned into the C-pB27 bait plasmid. doi:10.1371/journal.pone.0051555.g004

Article Snippet: For MYBBP1A, RelA/p65, PIAS1 and SIRT6 immunoprecipitates SIRT6 total cell lysate was incubated with following antibodies bound to protein G Agarose overnight at 4uC: polyclonal rabbit anti MYBBP1A (Bethyl Lab A301-328A, lot 301-328A-1), rabbit anti RelA/p65 (Bethyl Lab A301-824A), anti PIAS1 (Abcam ab32219), anti SIRT6 (Bethyl LabA302-451A).

Techniques: Sequencing, Mutagenesis, Amplification, Clone Assay, Binding Assay, Plasmid Preparation, Transformation Assay, Positive Control, Blocking Assay, Expressing, Construct, Control

Journal: PloS one

Article Title: Identification of novel interacting partners of Sirtuin6.

doi: 10.1371/journal.pone.0051555

Figure Lengend Snippet: Figure 5. Confirmation of the interaction of SIRT6 with MYBBP1A. HEK293 cells were transiently transfected with Flag-tagged SIRT6. Cell extracts were prepared and immunoprecipitated with an antibody to Flag followed by Western blotting with an antibody to MYBBP1A (lane 1 Flag IP). As a ‘‘no antibody’’ control the cell extract was immunoprecipitated with no anti-Flag antibody but with Protein G agarose alone (lane 2 control IP). As additional controls, cell extracts were also immunoprecipitated with anti-Flag antibody from cell extracts prepared from cells transfected with a control empty plasmid (lane 3 Flag IP control plasmid) or extracts were immunoprecipitated with anti-Flag from cell extracts prepared from untransfected cells (lane 4 Flag IP untransfected). doi:10.1371/journal.pone.0051555.g005

Article Snippet: For MYBBP1A, RelA/p65, PIAS1 and SIRT6 immunoprecipitates SIRT6 total cell lysate was incubated with following antibodies bound to protein G Agarose overnight at 4uC: polyclonal rabbit anti MYBBP1A (Bethyl Lab A301-328A, lot 301-328A-1), rabbit anti RelA/p65 (Bethyl Lab A301-824A), anti PIAS1 (Abcam ab32219), anti SIRT6 (Bethyl LabA302-451A).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Plasmid Preparation

Journal: PloS one

Article Title: Identification of novel interacting partners of Sirtuin6.

doi: 10.1371/journal.pone.0051555

Figure Lengend Snippet: Figure 6. PIAS1 SIRT6 co-immunoprecipitation. HEK293 cell extracts were prepared and immunoprecipitated with an antibody to PIAS1 (lane 1 aPIAS1 IP) or an irrelevant antibody (lane 2 Control IP) followed by Western blotting with anti-PIAS1 antibody. The PIAS1 band was seen to migrate just above the 62 kDa marker. HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 (lane 3) or the H133W mutant (lane 4) and immunoprecipitated with anti-Flag followed by Western blotting with anti-PIAS1 (a-Flag IP). In a separate experiment HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 and cell extracts were prepared and immunoprecip- itated with an anti-Flag antibody and analysed by Western blotting with anti-PIAS1 antibody (lanes 5–8). Lane 5 represents the total cell extract used in the immunoprecipitation (input). Lane 6 represents the anti- Flag immunoprecipitation eluted from the beads with Flag peptide (a- Flag IP). Lanes 7 represents the non-immunoprecipitated ‘‘flow through’’ material from the cell extract (FT) and lanes 8 represents the immunoprecipitated material eluted from the beads with SDS-PAGE sample buffer (a-Flag IP). doi:10.1371/journal.pone.0051555.g006

Article Snippet: For MYBBP1A, RelA/p65, PIAS1 and SIRT6 immunoprecipitates SIRT6 total cell lysate was incubated with following antibodies bound to protein G Agarose overnight at 4uC: polyclonal rabbit anti MYBBP1A (Bethyl Lab A301-328A, lot 301-328A-1), rabbit anti RelA/p65 (Bethyl Lab A301-824A), anti PIAS1 (Abcam ab32219), anti SIRT6 (Bethyl LabA302-451A).

Techniques: Immunoprecipitation, Control, Western Blot, Marker, Transfection, Mutagenesis, SDS Page

Journal: PloS one

Article Title: Identification of novel interacting partners of Sirtuin6.

doi: 10.1371/journal.pone.0051555

Figure Lengend Snippet: Figure 7. SMARCA5 SIRT6 co-immunoprecipitation. HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6. Cell extracts were prepared and immunoprecipitated with an irrelevant antibody (lane 1 Control IP) or an antibody to SMARCA5 (lane 2 aSMARCA5 IP). The SMARCA5 band was seen to migrate above the 98 kDa marker. In a separate experiment HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 and cell extracts were prepared and immunoprecipitated with an anti-Flag antibody and analysed by Western blotting with anti-SMARCA5 antibody (lanes 3–6). Lane 3 represents the total cell extract used in the immunoprecipitation (input). Lane 4 represents the anti-Flag immunoprecipitation eluted from the beads with Flag peptide (a-Flag IP). Lanes 5 represents the non-immunoprecipitated ‘‘flow through’’ material from the cell extract (FT) and lanes 6 represents the immunoprecipitated material eluted from the beads with SDS-PAGE sample buffer (a-Flag IP). doi:10.1371/journal.pone.0051555.g007

Article Snippet: For MYBBP1A, RelA/p65, PIAS1 and SIRT6 immunoprecipitates SIRT6 total cell lysate was incubated with following antibodies bound to protein G Agarose overnight at 4uC: polyclonal rabbit anti MYBBP1A (Bethyl Lab A301-328A, lot 301-328A-1), rabbit anti RelA/p65 (Bethyl Lab A301-824A), anti PIAS1 (Abcam ab32219), anti SIRT6 (Bethyl LabA302-451A).

Techniques: Immunoprecipitation, Transfection, Control, Marker, Western Blot, SDS Page

Journal: PloS one

Article Title: Identification of novel interacting partners of Sirtuin6.

doi: 10.1371/journal.pone.0051555

Figure Lengend Snippet: Figure 8. HEK293 cells were transiently transfected with Flag-tagged wild type SIRT6 and cell extracts were prepared and immunoprecipitated with a variety of antibodies and analysed by Coomassie staining the SDS-PAGE (lanes 1,2) or by Western blotting with anti-SIRT6 (lanes 3–11). Lanes 2 shows a stained SDS-PAGE of a HEK293 cell extract immunoprecipitated with anti-Flag (a-Flag IP) and lane 1 shows a control where the extract was omitted (no extract control). The three bands seen in lane 2 (a,b,c) were excised and sequenced by ms (Table 2). Lanes 3–11 are all anti-SIRT6 Western blots. Lane 3 shows a control immunoprecipitation where the anti-Flag antibody was omitted from the IP (no IP antibody control). Lane 4 shows the anti-Flag immunoprecipitate and lane 5 shows a sample of the total cell extract without immunoprecipitation. Lane 6 shows a sample (150 ng) of purified recombinant SIRT6 [17]. In a separate experiment and at higher exposure of the Western blot, lane 7 shows an anti-Flag IP, lane 8 shows an anti-SMARCA5 IP, lane 9 shows an anti-PIAS1 IP, lane 10 shows an anti-MYBBP1A IP and lane 11 shows an anti-SMARCA5 IP from H133W transfected cells. The figure is a composite of more than one experiment but represents data from three repeated experiments. doi:10.1371/journal.pone.0051555.g008

Article Snippet: For MYBBP1A, RelA/p65, PIAS1 and SIRT6 immunoprecipitates SIRT6 total cell lysate was incubated with following antibodies bound to protein G Agarose overnight at 4uC: polyclonal rabbit anti MYBBP1A (Bethyl Lab A301-328A, lot 301-328A-1), rabbit anti RelA/p65 (Bethyl Lab A301-824A), anti PIAS1 (Abcam ab32219), anti SIRT6 (Bethyl LabA302-451A).

Techniques: Transfection, Immunoprecipitation, Staining, SDS Page, Western Blot, Control, Purification, Recombinant

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 initially delays Delta16HER2-driven tumorigenesis, but then promotes a high tumor multiplicity. ( A ) Breeding scheme used to obtain Delta16HER2/SIRT6-OE female mice and a representative genotyping PCR. ( B ) Kaplan-Meir curves comparing the percentage of tumor-free mice between Delta16HER2 controls and Delta16HER2/SIRT6-OE mice (n = 17/group). ( C ) Representative post mortem pictures of Delta16HER2 and Delta16HER2/SIRT6-OE mice at 30 weeks of age (top panels), and relative H&E staining of the primary mammary tumor masses (bottom panels). Black arrows indicate tumor foci. ( D , E ) Tumor multiplicity and tumor growth curves of Delta16HER2 and Delta16HER2/SIRT6-OE mice (n = 17/group). ( F ) mRNA expression of Sirtuin1-7(Sirt1-7) and Delta16HER2 normalized to β-Actin mRNA level in tumors of 30-week-old Delta16HER2 controls and Delta16HER2/SIRT6-OE mice (n = 4/group). ( G ) Representative images of IHC staining for SIRT6 (brown) in tumors of 30-week-old Delta16HER2 and Delta16HER2/SIRT6-OE mice, respectively. Delta16HER2/SIRT6-OE tumors show a strong SIRT6 level and mainly localized into the nucleus. Scale bar, 25 mm. ( H , I ) Western blot analysis and relative quantification of total SIRT6 and phosphorylated SIRT6 (pSIRT6-Ser388) normalized to β-Actin protein level. pSIRT6-Ser388/total SIRT6 represents the ratio of β-Actin-normalized phosphorylated protein over the total SIRT6 protein. ( J ) Flow cytometry staining for Delta16HER2 and SIRT6 of primary cells (passage 4 in vitro) derived from tumors of Delta16HER2 and Delta16HER2/SIRT6-OE mice at 30 weeks of age. MFI, Median Fluorescence Intensity. The experiment was carried out in triplicates. In ( B ) ***p = 0.0008 (Log-rank test); in ( D , E ) **p < 0.01, ***p < 0.001 (two-way ANOVA followed by Sidak’s multiple comparisons test); in ( F , H , J ) *p < 0.05, **p < 0.01 (two-tailed unpaired t test). Error bars represent SD. See also Fig. .

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Staining, Expressing, Immunohistochemistry, Western Blot, Quantitative Proteomics, Flow Cytometry, In Vitro, Derivative Assay, Fluorescence, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 boosts tumor cell migration, invasion and lung metastasis in Delta16HER2/SIRT6-OE mice. ( A ) Representative pictures of H&E staining on lungs of Delta16HER2 and Delta16HER2/SIRT6-OE mice at 30 weeks of age (n = 7/group). Dashed black lines highlight the metastatic area in each picture. Quantification is represented as number of metastasis per mouse ( B ) and as metastasis area over total section area ( C ). Both number and area of metastasis were calculated as average over 2–6 consecutive tissue sections per mouse. ( D ) Soft agar assay performed on primary cells (passage 4 in vitro) derived from tumors of Delta16HER2 and Delta16HER2/SIRT6-OE mice at 30 weeks of age. Stereomicroscope images, 4X magnification (top) and 10X zoom (bottom). Number of colonies ( E ) were quantified using ImageJ software. Results are expressed as % of total number of seeded cells. The assay was performed in triplicates. ( F , G ) Transwell migration assay and quantification of Delta16HER2 and Delta16HER2/SIRT6-OE primary cells. Pore size 8.0 mm. Quantification is reported as percentage of migrating cells over the total number of seeded cells. In ( B , C , E , G ) *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired t test). Error bars represent SD.

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Migration, Staining, Soft Agar Assay, In Vitro, Derivative Assay, Software, Transwell Migration Assay, Pore Size, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 prevents tumor cells from Delta16HER2-induced G2/M arrest and senescence. ( A ) Ex vivo cell cycle analysis of cell suspensions derived from Delta16HER2 and Delta16HER2/SIRT6-OE tumors at 20 weeks of age (top panels) and at 30 weeks of age (bottom panels). DNA content was evaluated by flow cytometry using propidium iodide (PI) staining (n = 3) and then analyzed using Dean-Jett-Fox algorithm in FlowJo software. Percentages of cells in each cell cycle phase are summarized in each panel. ( B ) Immunoblot image and quantification of Cyclin D1 and Cyclin E levels normalized to β-Actin in tumors of either 20- (top) or 30-week-old (bottom) Delta16HER2 and Delta16HER2/SIRT6-OE mice (n = 4). ( C ) mRNA expression of Trp53 , Cdkn2a and Cdkn1a genes normalized to β-Actin mRNA level in tumors of 30-week-old Delta16HER2 controls and Delta16HER2/SIRT6-OE mice (n = 4/group). ( D ) Detection of senescence-associated β-galactosidase (SA-β-Gal) activity in frozen tumors of 30-week-old Delta16HER2 and Delta16HER2/SIRT6-OE (n = 3). Quantification is expressed as % of SA-β-Gal positive area (blue) with respect to the total section area. Scale bar, 25 mm. In ( B – D ) ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed unpaired t test). Error bars represent SD. See also Fig. .

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Ex Vivo, Cell Cycle Assay, Derivative Assay, Flow Cytometry, Staining, Software, Western Blot, Expressing, Activity Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 promotes stemness and self-renewal capacity of Delta16HER2 tumor cells. ( A – D ) Flow Cytometry analysis of CD44, CD24, OCT3/4, Notch1 and NANOG levels in primary tumor cells derived from 30-week-old Delta16HER2 and Delta16HER2/SIRT6-OE mice (n = 4). Quantification is expressed as median fluorescence intensity (MFI) ( A ) and as percentage of positive cells ( C and D ). ( E ) Representative images of mammosphere formation assay performed on primary tumor cells derived from 30-week-old Delta16HER2 and Delta16HER2/SIRT6-OE mice (n = 4, top panel). Quantification is represented for 2 serial cloning assays and is expressed as percentage of mammosphere forming units (MFU) with respect to the total number of seeded cells (bottom panel). See also Fig. . ( F ) Flow cytometry analysis of SIRT6 and HER2 expression in CAM6 cells stably transduced with pLENTI-Blank (empty vector) and pLENTI-SIRT6. ( G ) Mammosphere formation assay on CAM6-pLENTI-Blank and CAM6-pLENTI-SIRT6. Scale bar, 200 mm. ( H , I ) Quantification of mammosphere forming capacity (MFU %) and average sphere area of CAM6-pLENTI-Blank and CAM6-pLENTI-SIRT6 cells. Experiment was carried out in triplicate. ( J ) Immunofluorescence staining of OCT3/4 on CAM6-pLENTI-Blank and CAM6-pLENTI-SIRT6 mammospheres. DAPI is used for nuclei counterstaining. Magnification 40X. In ( A , C – E , H , I ) *p < 0.05, **p < 0.01, ***P < 0.001 (two-tailed unpaired t test). Error bars represent SEM.

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Flow Cytometry, Derivative Assay, Fluorescence, Tube Formation Assay, Cloning, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Immunofluorescence, Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6 is amplified in a subset of HER2-positive breast cancer patients and correlates with poor relapse-free-survival. ( A ) cBioPortal data summarizing the type and frequency of alteration of HER2 ( ERBB2 ) and SIRT6 genes in sequenced samples from publicly available invasive breast cancer studies (listed in the “study of origin” section). A total of 4860 and 4379 breast cancer patients were profiled for ERBB2 and SIRT6 alterations, respectively. ( B ) Co-occurrence/Mutual exclusivity analysis from cBioPortal indicating the number of patient samples that have amplification of HER2 (ERBB2) and SIRT6 alone, in both or neither genes. Odd ratio = neither*both/SIRT6 not ERBB2*ERBB2 not SIRT6. Log2 Odd Ratio > 1 indicates co-occurrence of HER2 and SIRT6 amplifications (p < 0.0001, Pearson correlation). ( C – E ) Kaplan–Meier plots from GOBO database using overall survival ( C ) and relapse-free survival (RFS) ( D , E ) as outcome in all HER2-enriched breast cancer ( D ) and in grade 3 HER2-enriched breast cancer ( E ). Data have been stratified into the two quantiles based on SIRT6 gene expression level (SIRT6_low, grey line and SIRT6_high, red line) using 10-year censoring as endpoint. ( F ) DNA microarray data from bc-GenExMiner showing SIRT6 expression in HER2- (n = 4068) and HER2 + (n = 680) breast cancer samples. ( G , H ) Kaplan–Meier plots from bc-GenExMiner using distant metastasis free survival (DMFS) and disease free survival (DFS) as outcomes in HER2-enriched breast cancer. Data have been stratified into the two quantiles based on SIRT6 gene expression level (SIRT6_low, purple line and SURT6_high, blue line). HR (hazard ratio) and 95% CI (confidence interval) are reported in the figure. In ( C , D , F , G ) *p = 0.02436, *p = 0.02143, *p = 0.0468, *p = 0.0222 (Log-rank test). In ( E ) *p = 0.0322 (Welch’s test). See also Figs. and .

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Amplification, Gene Expression, Microarray, Expressing

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: ChIP- and RNA-seq identify TBX3 as a SIRT6 target and a prognostic marker in HER2 + breast cancer. ( A ) Venn diagram representing the genes that are differentially expressed and differentially bound by H3K9ac (cutoff FDR ≤ 0.05) in tumors from Delta16HER2/SIRT6-OE versus Delta16HER2 controls (20 weeks of age, n = 2 for RNA-seq and n = 2–3 for ChIP-seq). ( B , C ) ChIP and RNA-seq tracks showing H3K9ac level and expression for Il12a and Tbx3 genes in Delta16HER2 (blue) versus Delta16HER2/SIRT6-OE (red) tumors (at 20 weeks of age). ( D ) Bee swarm plots computed in bc-GenExMiner showing the expression levels of IL12A and TBX3 in HER2–/ + breast cancers. ( E , F ) Kaplan–Meier plots from bc-GenExMiner using distant metastasis free survival (DMFS) and disease free survival (DFS) as outcomes in HER2-enriched breast cancer. Data have been stratified into the two quantiles based on IL12A gene expression level (( E ), IL12A_low, purple line and IL12A_high, blue line) and TBX3 gene expression level (( F ), TBX3_low, purple line and TBX3_high, blue line). HR (hazard ratio) and 95% CI (confidence interval) are reported in the figure. In ( D ) ****P < 0.0001 (Welch’s test). In ( E , F ) ns, p = 0.1124; ns, p = 0.7269; ***p = 0.0004, **p = 0.0075 (Log-rank test). See also Figs. and .

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: RNA Sequencing, Marker, ChIP-sequencing, Expressing, Gene Expression

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: SIRT6-OE induces loss of TBX3 and aggressiveness in both mouse and human models of Delta16HER2/HER2 + . ( A ) Representative pictures and ( B ) quantification for Tbx3 IHC in the indicated mouse tumors. ( C ) Immunoblot of Tbx3 in mouse tumors of the indicated genotype (20 weeks, n = 4 per group). ( D ) Representative pictures of the human BT474 cell line transfected with either pHIV-dTomato or pHIV-SIRT6-dTomato. ( E ) Real-time PCR for SIRT6 and TBX3 expression in sorted BT474 transfected as indicated. Not transfected HEK293 were used as control sample and TBP as housekeeping gene. ( F ) Representative pictures and ( G ) quantification of the Transwell migration assay of the human BT474 cell line transfected with either pHIV-dTomato or pHIV-SIRT6-dTomato. ( H ) Representative pictures and ( I ) number and area quantification of pHIV-dTomato and pHIV-SIRT6-dTomato BT474 mammospheres. MFU% = % of mammary forming units over total number of seeded cells. A.U . arbitrary unit. Bars indicate mean ± SD. In ( B ) ***p = 0.0009; ( C ) **p = 0.0077; ( D ) **p = 0.0014, ***p = 0.0003; ( G ) **p = 0.0077 and ( I ) statistics indicates unpaired two-tailed t test.

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Western Blot, Transfection, Real-time Polymerase Chain Reaction, Expressing, Control, Transwell Migration Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: Loss of TBX3 mimics SIRT6 -OE in vitro and predicts poor survival of HER2-positive breast cancer patients. ( A ) Immunoblot and quantification of TBX3 and GAPDH in lysates of BT474 cell line transfected with the indicated siRNA (48 h post transfection, n = 2 independent replicates). ( B ) Representative pictures and ( C ) quantification of the Transwell migration assay of the human BT474 cell line transfected as indicated (n = 5). ( D ) Representative pictures and ( E ) number and area quantification of siRNA control and siRNA TBX3 BT474 mammospheres (n = 3). MFU% = % of mammary forming units over total number of seeded cells. A.U . arbitrary unit. Bars indicate mean ± SD. See also Fig. . ( F ) Lollipop graph from cBioPortal showing mutations of the TBX3 gene in breast cancer patients (n = 338). Y axis represents the number of mutations while X axis indicates the correspondent amino acid position. OncoKB tracks in blue indicate those mutations that are likely-oncogenic and likely loss-of-function. Types of mutation are color-coded in the figure legend. ( G ) Summary of total number and frequency of TBX3 mutations in all, HER2-WT and HER2 amplified breast cancer subsets. Pie charts indicate the frequency of the different types of TBX3 mutations over total number of TBX3 mutations in each subset. ( H ) Kaplan–Meier plots from cBioPortal showing the overall survival of patients with HER2 amplified alone (n = 271), and those with concomitant SIRT6 amplification (n = 8) or TBX3 loss-of-function mutations (n = 20) . P = 0.0007 (Log-rank Mantel-Cox test). In ( A ) **p = 0.0047, ( C ) *p = 0.0282, ( E ) **p = 0.0051 and ns, p = 0.2106 indicates unpaired two-tailed t test.

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: In Vitro, Western Blot, Transfection, Transwell Migration Assay, Control, Mutagenesis, Amplification, Two Tailed Test

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: qRT-PCR primer list.

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: Sequencing, Amplification

Journal: Scientific Reports

Article Title: SIRT6 promotes metastasis and relapse in HER2-positive breast cancer

doi: 10.1038/s41598-023-49199-7

Figure Lengend Snippet: Summary of used antibodies.

Article Snippet: Phospho-SIRT6 , Rabbit polyclonal anti-phospho sirt6 (ser338) , WB , 1:1000 , Biorbyt.

Techniques: