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: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hepatic SIRT6 Modulates Transcriptional Activities of FXR to Alleviate Acetaminophen-induced Hepatotoxicity

doi: 10.1016/j.jcmgh.2022.04.011

Figure Lengend Snippet: Hepatic SIRT6 deficiency deteriorates APAP overdose-induced acute liver injury. Occurrences at 12 hours following APAP injection. ( A ) Serum ALT and AST levels of SIRT6-LKO and control mice after APAP injection; ( B ) SIRT6-LKO mice displayed higher ratio of liver weight relative to body weight after APAP injection; ( C and D ) H&E and TUNEL assay showed deteriorated centrilobular necrosis and liver cell apoptosis in SIRT6-LKO mice after APAP administration; ( E ) Western blotting indicated an increased rate of hepatic Bax relative to Bcl2 expression in SIRT6-LKO mice after APAP administration; ( F ) ELISA assay showed increased NAPQI levels in APAP-treated SIRT6-LKO mice; ( G ) Hepatic SIRT6 knockout leads to change of genes involved in APAP metabolism; ( H ) Hepatic SIRT6 deficiency decreased the expression of anti-oxidative genes including SOD2 and Gclc; ( I ) Hepatic GSH and SOD levels were reduced in SIRT6-LKO mice after APAP treatment; ( J ) Hepatic ROS levels were increased in SIRT6-LKO mice after APAP treatment; ( K and L ) SIRT6 deletion leads to elevated ROS generation and cell apoptosis in MPHs. Data are means ± standard error of the mean; n = 6 to 8 mice/group. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001. Similar results were obtained in 3 independent experiments.

Article Snippet: Western blot assays were performed using antibodies specific for rabbit anti-Bcl2, rabbit anti-Bax, rabbit anti-SIRT6, mouse anti-β-actin (Abclonal, Wuhan, China), and rabbit anti-NF-κBp65 (Cell Signaling Technology, Danvers, MA).

Techniques: Injection, TUNEL Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hepatic SIRT6 Modulates Transcriptional Activities of FXR to Alleviate Acetaminophen-induced Hepatotoxicity

doi: 10.1016/j.jcmgh.2022.04.011

Figure Lengend Snippet: SIRT6 overexpression prominently improves APAP overdose-induced acute liver injury. Occurrences at 12 hours following APAP injection. ( A ) Quantitative polymerase chain reaction data showed a hepatic overexpression of SIRT6 in Ad-SIRT6-infected mice; ( B ) SIRT6 overexpression increased the survival rate after indicated time of APAP injection; ( C ) SIRT6 overexpression decreased serum ALT and AST levels after APAP treatment; ( D ) SIRT6 overexpression reduced the ratio of liver weight relative to body weight in APAP-injected mice; ( E and F ) APAP-induced centrilobular necrosis ( E ) and liver cell apoptosis ( F ) were improved in Ad-SIRT6-infected mice; ( G ) SIRT6 overexpression decreased the ratio of hepatic Bax relative to Bcl2 expression; ( H ) ELISA assay showed a decrease of NAPQI levels in Ad-SIRT6-infected mice; ( I and J ) Hepatic GSH ( I ) and ROS ( J ) levels were decreased in Ad-SIRT6-infected mice after APAP treatment; ( K and L ) Hepatic SIRT6 overexpression altered genes involved in APAP metabolism; ( M ) Hepatic SIRT6 overexpression increased anti-oxidative genes including SOD2 and Gclc. Data are means ± standard error of the mean; n = 6 to 8 mice/group. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001. Similar results were obtained in 3 independent experiments.

Article Snippet: Western blot assays were performed using antibodies specific for rabbit anti-Bcl2, rabbit anti-Bax, rabbit anti-SIRT6, mouse anti-β-actin (Abclonal, Wuhan, China), and rabbit anti-NF-κBp65 (Cell Signaling Technology, Danvers, MA).

Techniques: Over Expression, Injection, Real-time Polymerase Chain Reaction, Infection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hepatic SIRT6 Modulates Transcriptional Activities of FXR to Alleviate Acetaminophen-induced Hepatotoxicity

doi: 10.1016/j.jcmgh.2022.04.011

Figure Lengend Snippet: Mutant SIRT6 deacetylase domain fails to alter APAP-induced liver injury. Occurrences at 12 hours following APAP injection, ( A ) liver percentage and ( B ) serum ALT and AST levels in Ad-Ctrl- or Ad-Sirt6 (H133Y)-infected mice after APAP injection; ( C and D ) H&E and TUNEL assay showed no change of centrilobular necrosis and liver cell apoptosis in Ad-Sirt6 (H133Y) mice after APAP administration. ( E ) Western blotting indicated no change of hepatic Bax relative to Bcl2 expression in Ad-Sirt6 (H133Y) mice after APAP administration. ( F–H ) No change of GSH levels ( F ), hepatic antioxidantive genes expression ( G ), NAPQI and APAP-metabolism related genes expression ( H ). ( I and J ) Mutant SIRT6 deacetylase domain failed to alter change the expression of hepatic F4/80 and serum inflammatory cytokines levels. Data are means ± standard error of the mean; n = 6 to 8 /group. ∗ P < .05, ∗∗ P < .01.

Article Snippet: Western blot assays were performed using antibodies specific for rabbit anti-Bcl2, rabbit anti-Bax, rabbit anti-SIRT6, mouse anti-β-actin (Abclonal, Wuhan, China), and rabbit anti-NF-κBp65 (Cell Signaling Technology, Danvers, MA).

Techniques: Mutagenesis, Histone Deacetylase Assay, Injection, Infection, TUNEL Assay, Western Blot, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hepatic SIRT6 Modulates Transcriptional Activities of FXR to Alleviate Acetaminophen-induced Hepatotoxicity

doi: 10.1016/j.jcmgh.2022.04.011

Figure Lengend Snippet: Pharmacological activation of SIRT6 improves APAP overdose-induced acute liver injury. Occurrences at 12 hours following APAP injection. ( A ) MDL-800 treatment decreased hepatic centrilobular necrosis levels after APAP treatment; ( B ) MDL-800 treatment reduced serum ALT and AST levels after APAP treatment; ( C and D ) MDL-800 treatment decreased liver cell apoptosis ( C ) and the ratio of Bax relative to Bcl 2 ; ( E ) MDL-800 administration increased hepatic SOD2 expression and decreased hepatic ROS levels after APAP treatment; ( F ) MDL-800 treatment decreased nuclear NF-κBp65 protein levels after APAP treatment; ( G and H ) MDL-800 treatment decreased pro-inflammatory cytokine release ( G ) and gene expression ( H ) . Data are means ± standard error of the mean; n = 6 to 8 mice/group. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001. Similar results were obtained in 3 independent experiments.

Article Snippet: Western blot assays were performed using antibodies specific for rabbit anti-Bcl2, rabbit anti-Bax, rabbit anti-SIRT6, mouse anti-β-actin (Abclonal, Wuhan, China), and rabbit anti-NF-κBp65 (Cell Signaling Technology, Danvers, MA).

Techniques: Activation Assay, Injection, Expressing