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    Cell Signaling Technology Inc etdrs letters 60 5 11 0 60 8 10 6 62 mean cst
    Etdrs Letters 60 5 11 0 60 8 10 6 62 Mean Cst, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    etdrs letters 60 5 11 0 60 8 10 6 62 mean cst  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc etdrs letters 60 5 11 0 60 8 10 6 62 mean cst
    Etdrs Letters 60 5 11 0 60 8 10 6 62 Mean Cst, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ddb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ddb2
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    Cell Signaling Technology Inc sc ddb2
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    Cell Signaling Technology Inc ddb2
    ERα mediates chemotherapy-induced <t>DDB2</t> gene transcription (A and B) Basal mRNA (A) and protein (B) levels of DDB2 were higher in ER-positive than ER-negative breast cancer cell lines, regardless of their TP53 genetic status. (C) Silencing of ERα by two independent shRNAs suppressed DDB2 protein and mRNA expression. Reduction of both S118-phosphorylated and total ERα revealed the inhibition of ERα activity. (D) ER response elements (EREs) and p53 RE (p53RE) motifs on the DDB2 promoter and the luciferase reporter constructs driven by different lengths of the DDB2 promoter were illustrated (vermilion, red, and pink: EREs; orange: p53RE). (E) Treatment with carboplatin (50 μM) increased the luciferase activity driven by the DDB2 promoter containing both p53RE and EREs in mutp53-expressing T-47D cancer cells. Firefly luciferase activity was normalized with β-gal activity. (F) Silencing of ERα decreased the carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells, and firefly luciferase activity was normalized with β-gal activity. Data in (A), (C), (E), and (F) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.
    Ddb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "ERα determines the chemo-resistant function of mutant p53 involving the switch between lincRNA-p21 and DDB2 expressions"

    Article Title: ERα determines the chemo-resistant function of mutant p53 involving the switch between lincRNA-p21 and DDB2 expressions

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2021.07.022

    ERα mediates chemotherapy-induced DDB2 gene transcription (A and B) Basal mRNA (A) and protein (B) levels of DDB2 were higher in ER-positive than ER-negative breast cancer cell lines, regardless of their TP53 genetic status. (C) Silencing of ERα by two independent shRNAs suppressed DDB2 protein and mRNA expression. Reduction of both S118-phosphorylated and total ERα revealed the inhibition of ERα activity. (D) ER response elements (EREs) and p53 RE (p53RE) motifs on the DDB2 promoter and the luciferase reporter constructs driven by different lengths of the DDB2 promoter were illustrated (vermilion, red, and pink: EREs; orange: p53RE). (E) Treatment with carboplatin (50 μM) increased the luciferase activity driven by the DDB2 promoter containing both p53RE and EREs in mutp53-expressing T-47D cancer cells. Firefly luciferase activity was normalized with β-gal activity. (F) Silencing of ERα decreased the carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells, and firefly luciferase activity was normalized with β-gal activity. Data in (A), (C), (E), and (F) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.
    Figure Legend Snippet: ERα mediates chemotherapy-induced DDB2 gene transcription (A and B) Basal mRNA (A) and protein (B) levels of DDB2 were higher in ER-positive than ER-negative breast cancer cell lines, regardless of their TP53 genetic status. (C) Silencing of ERα by two independent shRNAs suppressed DDB2 protein and mRNA expression. Reduction of both S118-phosphorylated and total ERα revealed the inhibition of ERα activity. (D) ER response elements (EREs) and p53 RE (p53RE) motifs on the DDB2 promoter and the luciferase reporter constructs driven by different lengths of the DDB2 promoter were illustrated (vermilion, red, and pink: EREs; orange: p53RE). (E) Treatment with carboplatin (50 μM) increased the luciferase activity driven by the DDB2 promoter containing both p53RE and EREs in mutp53-expressing T-47D cancer cells. Firefly luciferase activity was normalized with β-gal activity. (F) Silencing of ERα decreased the carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells, and firefly luciferase activity was normalized with β-gal activity. Data in (A), (C), (E), and (F) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.

    Techniques Used: Expressing, Inhibition, Activity Assay, Luciferase, Construct

    ERα cooperates with mutp53 to upregulate DDB2 gene transcription and mediates chemoresistance in ER-positive breast cancer cells (A) Silencing of p53 decreased the carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells. (B) Deletions of EREs, but not p53RE, reduced carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells. Firefly luciferase activity was normalized with β-gal activity in (A) and (B). (C) Carboplatin (50 μM) induced chromatin-binding affinity of both ERα and mutp53 preferentially on ERE#1 of the DDB2 promoter in T-47D cancer cells in the ChIP assay. (D) Silencing of ERα reduced p53 activity, as evidenced by the acetylation at K382 in T-47D and BT-474 cancer cells. (E) Chemotherapy (50 μM carboplatin or 0.5 μM doxorubicin) induced the protein interaction between p53 and ERα in the coimmunoprecipitation (coIP) assay. (F) Ectopic co-expression of ERα and p53 mutants (R280K and R273H) synergistically enhanced DDB2 expression in HEK293T cells. (G) Silencing of DDB2 by two independent shRNAs enhanced carboplatin (50 μM)-induced DNA damage in the comet assay. The tail moment and tail-length index were calculated from images by Comet Assay III analysis. Data in (A−C) and (G) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.
    Figure Legend Snippet: ERα cooperates with mutp53 to upregulate DDB2 gene transcription and mediates chemoresistance in ER-positive breast cancer cells (A) Silencing of p53 decreased the carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells. (B) Deletions of EREs, but not p53RE, reduced carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells. Firefly luciferase activity was normalized with β-gal activity in (A) and (B). (C) Carboplatin (50 μM) induced chromatin-binding affinity of both ERα and mutp53 preferentially on ERE#1 of the DDB2 promoter in T-47D cancer cells in the ChIP assay. (D) Silencing of ERα reduced p53 activity, as evidenced by the acetylation at K382 in T-47D and BT-474 cancer cells. (E) Chemotherapy (50 μM carboplatin or 0.5 μM doxorubicin) induced the protein interaction between p53 and ERα in the coimmunoprecipitation (coIP) assay. (F) Ectopic co-expression of ERα and p53 mutants (R280K and R273H) synergistically enhanced DDB2 expression in HEK293T cells. (G) Silencing of DDB2 by two independent shRNAs enhanced carboplatin (50 μM)-induced DNA damage in the comet assay. The tail moment and tail-length index were calculated from images by Comet Assay III analysis. Data in (A−C) and (G) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.

    Techniques Used: Activity Assay, Luciferase, Binding Assay, Co-Immunoprecipitation Assay, Expressing, Single Cell Gel Electrophoresis

    ERα hijacks mutp53 from the G-quadruplex DNA (GQ) structure of the lincRNA-p21 promoter to EREs of the DDB2 promoter (A) Illustration of the predicted non-B structure and p53RE on the lincRNA-p21 promoter and two luciferase-reporter constructs driven by lincRNA-p21 promoters containing different motifs. (B and C) GQ motif is required for mutp53-increased lincRNA-p21 promoter activity, and firefly luciferase activity was normalized with β-gal activity. (D) The binding efficacy of endogenous mutp53 on different motifs of the lincRNA-p21 promoter in response to chemotherapies (50 μM carboplatin and 0.5 μM doxorubicin) in the ChIP assay. (E and F) Carboplatin (50 μM) and two GQ stabilizers (1 μM NMM and 5 μM auramine) increased the binding of mutp53 to the GQ motif of the 5′-biotinylated lincRNA-p21 promoters in the in vitro pull-down assay. (G) GQ stabilizers increased lincRNA-p21 expression in MDA-MB-231 cancer cells. (H) Silencing of ERα switched the chromatin-binding activity of mutp53 from EREs of the DDB2 promoter (left) to the non-B DNA motifs of the lincRNA-p21 promoter (right) in ER-positive/p53 L194F T-47D cancer cells. (I) The inversed correlation between the ex vivo induction of lincRNA-p21 and DDB2 expression by carboplatin (50 μM) treatments in human primary breast cancer tissues in an ER status-dependent manner. Data in (B−D), (G), and (H) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.
    Figure Legend Snippet: ERα hijacks mutp53 from the G-quadruplex DNA (GQ) structure of the lincRNA-p21 promoter to EREs of the DDB2 promoter (A) Illustration of the predicted non-B structure and p53RE on the lincRNA-p21 promoter and two luciferase-reporter constructs driven by lincRNA-p21 promoters containing different motifs. (B and C) GQ motif is required for mutp53-increased lincRNA-p21 promoter activity, and firefly luciferase activity was normalized with β-gal activity. (D) The binding efficacy of endogenous mutp53 on different motifs of the lincRNA-p21 promoter in response to chemotherapies (50 μM carboplatin and 0.5 μM doxorubicin) in the ChIP assay. (E and F) Carboplatin (50 μM) and two GQ stabilizers (1 μM NMM and 5 μM auramine) increased the binding of mutp53 to the GQ motif of the 5′-biotinylated lincRNA-p21 promoters in the in vitro pull-down assay. (G) GQ stabilizers increased lincRNA-p21 expression in MDA-MB-231 cancer cells. (H) Silencing of ERα switched the chromatin-binding activity of mutp53 from EREs of the DDB2 promoter (left) to the non-B DNA motifs of the lincRNA-p21 promoter (right) in ER-positive/p53 L194F T-47D cancer cells. (I) The inversed correlation between the ex vivo induction of lincRNA-p21 and DDB2 expression by carboplatin (50 μM) treatments in human primary breast cancer tissues in an ER status-dependent manner. Data in (B−D), (G), and (H) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.

    Techniques Used: Luciferase, Construct, Activity Assay, Binding Assay, In Vitro, Pull Down Assay, Expressing, Ex Vivo

    lincRNA-p21 reduces the growth and chemoresistance of the ER-positive tumor in vivo (A) The T-47D#Tet-On-LincRNA-p21 stable clone was treated with or without tetracycline (10 μg/mL), and the expressions of lincRNA-p21 and DDB2 were detected by quantitative real-time PCR analysis and western blot assay, respectively. Data were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test. (B) Illustration of the treatment timeline in the tumor-xenograft mouse model (yellow arrow: the starting point for tetracycline administration [0.2 mg/mL]; red arrow: the points for the intraperitoneal injection with doxorubicin [2.5 mg/kg]). (C and D) The tumor growth rate (C) and size at the end point (D) in four groups of these mice. Data were representative of n = 3 in every group and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test. (E) The expressions of lincRNA-p21, DDB2, Ki67, and cleaved caspase-3 in the tumor tissues were examined in the in situ hybridization and immunohistochemistry assays.
    Figure Legend Snippet: lincRNA-p21 reduces the growth and chemoresistance of the ER-positive tumor in vivo (A) The T-47D#Tet-On-LincRNA-p21 stable clone was treated with or without tetracycline (10 μg/mL), and the expressions of lincRNA-p21 and DDB2 were detected by quantitative real-time PCR analysis and western blot assay, respectively. Data were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test. (B) Illustration of the treatment timeline in the tumor-xenograft mouse model (yellow arrow: the starting point for tetracycline administration [0.2 mg/mL]; red arrow: the points for the intraperitoneal injection with doxorubicin [2.5 mg/kg]). (C and D) The tumor growth rate (C) and size at the end point (D) in four groups of these mice. Data were representative of n = 3 in every group and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test. (E) The expressions of lincRNA-p21, DDB2, Ki67, and cleaved caspase-3 in the tumor tissues were examined in the in situ hybridization and immunohistochemistry assays.

    Techniques Used: In Vivo, Stable Transfection, Real-time Polymerase Chain Reaction, Western Blot, Injection, In Situ Hybridization, Immunohistochemistry

    The proposed model of ERα/mutp53 mediated DDB2 and lincRNA-p21 in contributing to chemoresistance The proposed model illustrates how ERα determined chemoresistance of breast cancer by disrupting the balance between mutp53-dependent DDB2 and lincRNA-p21 transcriptions. In the ER-negative cancer cells (left), lincRNA-p21 , transcribed by mutp53 in a G-quadruplex of non-B structure-dependent fashion, mediate apoptosis for chemosensitivity. In the ER-positive cancer cells (right), however, ERα switches mutp53 to preferentially mediate DDB2 transcription via targeting its EREs and thereby reduces lincRNA-p21 expression, conferring chemoresistance (p53∗, mutp53).
    Figure Legend Snippet: The proposed model of ERα/mutp53 mediated DDB2 and lincRNA-p21 in contributing to chemoresistance The proposed model illustrates how ERα determined chemoresistance of breast cancer by disrupting the balance between mutp53-dependent DDB2 and lincRNA-p21 transcriptions. In the ER-negative cancer cells (left), lincRNA-p21 , transcribed by mutp53 in a G-quadruplex of non-B structure-dependent fashion, mediate apoptosis for chemosensitivity. In the ER-positive cancer cells (right), however, ERα switches mutp53 to preferentially mediate DDB2 transcription via targeting its EREs and thereby reduces lincRNA-p21 expression, conferring chemoresistance (p53∗, mutp53).

    Techniques Used: Expressing

    sc ddb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc sc ddb2
    Sc Ddb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti ddb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti ddb2
    USP44 deubiquitinates <t>DDB2</t> following UVC exposure. (A,B) MEFs of the indicated genotype were exposed to 30 J/m 2 UVC and samples were collected for immunoblot at the indicated times. The doublet bands were consistently observed in these mouse cells, and were both quantitated to represent DDB2 using imageJ. The graph represents the mean ± SEM for three independent MEF lines. “*” denotes p < 0.05. (C) MEFs were transduced with the indicated constructs and subjected to immunoprecipitation for the FLAG epitope and probed as indicated. (D) Purified CRL-DDB2 was auto-ubiquitinated by the addition of UbE1, UBCH5a, and ubiquitin, and subsequently incubated with either wild type or catalytic mutant (C281A) immunopurified USP44 as indicated. Rxn = reaction.
    Anti Ddb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "USP44 Stabilizes DDB2 to Facilitate Nucleotide Excision Repair and Prevent Tumors"

    Article Title: USP44 Stabilizes DDB2 to Facilitate Nucleotide Excision Repair and Prevent Tumors

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.663411

    USP44 deubiquitinates DDB2 following UVC exposure. (A,B) MEFs of the indicated genotype were exposed to 30 J/m 2 UVC and samples were collected for immunoblot at the indicated times. The doublet bands were consistently observed in these mouse cells, and were both quantitated to represent DDB2 using imageJ. The graph represents the mean ± SEM for three independent MEF lines. “*” denotes p < 0.05. (C) MEFs were transduced with the indicated constructs and subjected to immunoprecipitation for the FLAG epitope and probed as indicated. (D) Purified CRL-DDB2 was auto-ubiquitinated by the addition of UbE1, UBCH5a, and ubiquitin, and subsequently incubated with either wild type or catalytic mutant (C281A) immunopurified USP44 as indicated. Rxn = reaction.
    Figure Legend Snippet: USP44 deubiquitinates DDB2 following UVC exposure. (A,B) MEFs of the indicated genotype were exposed to 30 J/m 2 UVC and samples were collected for immunoblot at the indicated times. The doublet bands were consistently observed in these mouse cells, and were both quantitated to represent DDB2 using imageJ. The graph represents the mean ± SEM for three independent MEF lines. “*” denotes p < 0.05. (C) MEFs were transduced with the indicated constructs and subjected to immunoprecipitation for the FLAG epitope and probed as indicated. (D) Purified CRL-DDB2 was auto-ubiquitinated by the addition of UbE1, UBCH5a, and ubiquitin, and subsequently incubated with either wild type or catalytic mutant (C281A) immunopurified USP44 as indicated. Rxn = reaction.

    Techniques Used: Western Blot, Transduction, Construct, Immunoprecipitation, FLAG-tag, Purification, Incubation, Mutagenesis

    Incomplete CPD repair in Usp44 null cells associated with inadequate DDB2 recruitment to sites of damage. (A,B) VH10 cells expressing DDB2-GFP were transfected with control or USP44 targeting siRNA and then locally irradiated. The DDB-GFP fluorescence was monitored over time using live-cell confocal imaging and quantified to pre-damage intensity set at 100. The graph represents the mean ± SEM for 31–36 cells per condition. (C) MEFs of the indicated genotypes were transduced with wild-type, catalytic mutant (USP44 C I ; C281A), centrin-binding deficient (USP44 C BM ; W162A) USP44, or DDB2 as indicated. The cells were exposed to UVC (10 J/m 2 ) and CPD levels were monitored at the indicated times. The graph represents the means of three independent experiments for each condition.
    Figure Legend Snippet: Incomplete CPD repair in Usp44 null cells associated with inadequate DDB2 recruitment to sites of damage. (A,B) VH10 cells expressing DDB2-GFP were transfected with control or USP44 targeting siRNA and then locally irradiated. The DDB-GFP fluorescence was monitored over time using live-cell confocal imaging and quantified to pre-damage intensity set at 100. The graph represents the mean ± SEM for 31–36 cells per condition. (C) MEFs of the indicated genotypes were transduced with wild-type, catalytic mutant (USP44 C I ; C281A), centrin-binding deficient (USP44 C BM ; W162A) USP44, or DDB2 as indicated. The cells were exposed to UVC (10 J/m 2 ) and CPD levels were monitored at the indicated times. The graph represents the means of three independent experiments for each condition.

    Techniques Used: Expressing, Transfection, Irradiation, Fluorescence, Imaging, Transduction, Mutagenesis, Binding Assay

    ddb2 ab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ddb2 ab
    Ddb2 Ab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti ddb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti ddb2
    In response to UV irradiation, SIRT6 interacts with <t>DDB2.</t> ( A ) SIRT6 fails to stimulate NER in XPC-depleted HCA2-hTERT cells. A control vector or vector encoding SIRT6 was cotransfected with UVC-treated pmax-GFP and pDsRed2-N1 into control and XPC-depleted HCA2-hTERT cells. At 72 h post transfection, the cells were harvested for FACS analysis. Error bars represent the s.d. *** P < 0.001, n.s., not significant. (B, C) SIRT6 interacts with DDB2 in vivo . HEK293 cells were transfected with Flag-tagged SIRT6. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the Flag tag, followed by western blot analysis with the indicated antibodies ( B ). HEK293 cells were transfected with HA-tagged DDB2. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the HA tag, followed by western blot analysis ( C ). ( D ) SIRT6 interacts with DDB2 in vitro . Five micrograms of recombinant His-DDB2 and GST or GST-SIRT6 together with 30 μl GST-agarose resin were incubated with GST or GST-SIRT6 in IP buffer for 6 h at 4°C. Western blot analysis was performed with the indicated antibodies. ( E ) A schematic representation of the SIRT6 fragments used in this study is shown. ( F ) A control vector or vectors encoding full-length SIRT6-Flag, SIRT6 ΔC-Flag (1-271 aa), SIRT6 ΔN-Flag (49–355 aa) or the deacetylase core fragment SIRT6 ΔNΔC-Flag (49–271 aa) were cotransfected with DDB2-GFP into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with GFP-Trap (Chromotek), followed by western blot analysis. ( G ) A schematic representation of the DDB2 fragments used in this study is shown. ( H ) Vectors encoding full-length DDB2-GFP, DDB2 fragment 1-GFP (1–100 aa), or DDB2 fragment 2-GFP (101–427 aa) were cotransfected with a SIRT6-Flag-expressing vector into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis. ( I ) SIRT6 fails to stimulate NER in DDB2-depleted HCA2-hTERT cells. HCA2-hTERT cells were transfected with control siRNA or DDB2-specific siRNA twice over a 48-h interval. Afterwards, the HCA2-hTERT cells were transfected with a control vector or a vector expressing SIRT6 and UVC-treated pmax-GFP together with pDsRed2-N1. On day 3 post transfection, the cells were harvested for FACS analysis. Depletion of DDB2 from fibroblasts was confirmed by western blot analysis. Error bars represent the s.d. ** P < 0.01, n.s., not significant.
    Anti Ddb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The deacetylase SIRT6 promotes the repair of UV-induced DNA damage by targeting DDB2"

    Article Title: The deacetylase SIRT6 promotes the repair of UV-induced DNA damage by targeting DDB2

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa661

    In response to UV irradiation, SIRT6 interacts with DDB2. ( A ) SIRT6 fails to stimulate NER in XPC-depleted HCA2-hTERT cells. A control vector or vector encoding SIRT6 was cotransfected with UVC-treated pmax-GFP and pDsRed2-N1 into control and XPC-depleted HCA2-hTERT cells. At 72 h post transfection, the cells were harvested for FACS analysis. Error bars represent the s.d. *** P < 0.001, n.s., not significant. (B, C) SIRT6 interacts with DDB2 in vivo . HEK293 cells were transfected with Flag-tagged SIRT6. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the Flag tag, followed by western blot analysis with the indicated antibodies ( B ). HEK293 cells were transfected with HA-tagged DDB2. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the HA tag, followed by western blot analysis ( C ). ( D ) SIRT6 interacts with DDB2 in vitro . Five micrograms of recombinant His-DDB2 and GST or GST-SIRT6 together with 30 μl GST-agarose resin were incubated with GST or GST-SIRT6 in IP buffer for 6 h at 4°C. Western blot analysis was performed with the indicated antibodies. ( E ) A schematic representation of the SIRT6 fragments used in this study is shown. ( F ) A control vector or vectors encoding full-length SIRT6-Flag, SIRT6 ΔC-Flag (1-271 aa), SIRT6 ΔN-Flag (49–355 aa) or the deacetylase core fragment SIRT6 ΔNΔC-Flag (49–271 aa) were cotransfected with DDB2-GFP into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with GFP-Trap (Chromotek), followed by western blot analysis. ( G ) A schematic representation of the DDB2 fragments used in this study is shown. ( H ) Vectors encoding full-length DDB2-GFP, DDB2 fragment 1-GFP (1–100 aa), or DDB2 fragment 2-GFP (101–427 aa) were cotransfected with a SIRT6-Flag-expressing vector into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis. ( I ) SIRT6 fails to stimulate NER in DDB2-depleted HCA2-hTERT cells. HCA2-hTERT cells were transfected with control siRNA or DDB2-specific siRNA twice over a 48-h interval. Afterwards, the HCA2-hTERT cells were transfected with a control vector or a vector expressing SIRT6 and UVC-treated pmax-GFP together with pDsRed2-N1. On day 3 post transfection, the cells were harvested for FACS analysis. Depletion of DDB2 from fibroblasts was confirmed by western blot analysis. Error bars represent the s.d. ** P < 0.01, n.s., not significant.
    Figure Legend Snippet: In response to UV irradiation, SIRT6 interacts with DDB2. ( A ) SIRT6 fails to stimulate NER in XPC-depleted HCA2-hTERT cells. A control vector or vector encoding SIRT6 was cotransfected with UVC-treated pmax-GFP and pDsRed2-N1 into control and XPC-depleted HCA2-hTERT cells. At 72 h post transfection, the cells were harvested for FACS analysis. Error bars represent the s.d. *** P < 0.001, n.s., not significant. (B, C) SIRT6 interacts with DDB2 in vivo . HEK293 cells were transfected with Flag-tagged SIRT6. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the Flag tag, followed by western blot analysis with the indicated antibodies ( B ). HEK293 cells were transfected with HA-tagged DDB2. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the HA tag, followed by western blot analysis ( C ). ( D ) SIRT6 interacts with DDB2 in vitro . Five micrograms of recombinant His-DDB2 and GST or GST-SIRT6 together with 30 μl GST-agarose resin were incubated with GST or GST-SIRT6 in IP buffer for 6 h at 4°C. Western blot analysis was performed with the indicated antibodies. ( E ) A schematic representation of the SIRT6 fragments used in this study is shown. ( F ) A control vector or vectors encoding full-length SIRT6-Flag, SIRT6 ΔC-Flag (1-271 aa), SIRT6 ΔN-Flag (49–355 aa) or the deacetylase core fragment SIRT6 ΔNΔC-Flag (49–271 aa) were cotransfected with DDB2-GFP into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with GFP-Trap (Chromotek), followed by western blot analysis. ( G ) A schematic representation of the DDB2 fragments used in this study is shown. ( H ) Vectors encoding full-length DDB2-GFP, DDB2 fragment 1-GFP (1–100 aa), or DDB2 fragment 2-GFP (101–427 aa) were cotransfected with a SIRT6-Flag-expressing vector into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis. ( I ) SIRT6 fails to stimulate NER in DDB2-depleted HCA2-hTERT cells. HCA2-hTERT cells were transfected with control siRNA or DDB2-specific siRNA twice over a 48-h interval. Afterwards, the HCA2-hTERT cells were transfected with a control vector or a vector expressing SIRT6 and UVC-treated pmax-GFP together with pDsRed2-N1. On day 3 post transfection, the cells were harvested for FACS analysis. Depletion of DDB2 from fibroblasts was confirmed by western blot analysis. Error bars represent the s.d. ** P < 0.01, n.s., not significant.

    Techniques Used: Irradiation, Plasmid Preparation, Transfection, In Vivo, Immunoprecipitation, FLAG-tag, Western Blot, In Vitro, Recombinant, Incubation, Histone Deacetylase Assay, Expressing

    SIRT6 deacetylates DDB2 in response to UV irradiation. ( A ) The interaction between SIRT6 and DDB2 is enhanced in response to UVC irradiation. HEK293 cells with stable Flag-tagged SIRT6 integration were transfected with DDB2-GFP and treated with or without UVC (20 J/m 2 ). Then, cells were harvested at the indicated time points. Cell lysates were immunoprecipitated with an anti-Flag antibody, followed by western blot analysis with the indicated antibodies. IPed, immunoprecipitated. ( B ) DDB2 is a target of the deacetylase SIRT6. HEK293 cells were cotransfected with plasmids encoding DDB2-GFP and SIRT6-Flag. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against acetylated lysines (AcK), followed by western blot analysis with the indicated antibodies. ( C ) DDB2 is deacetylated by SIRT6 in vitro . A recombinant DDB2-GFP protein (5 μg) and a SIRT6 WT or H133Y mutant protein (5 μg) purified from HEK293 cells were incubated to allow the deacetylation reaction to occur in HDAC buffer (50 mM Tris-HCl pH 9.0, 4 mM MgCl 2 , 50 mM NaCl, 0.2 mM DTT and 1 mM NAD+) at 30°C for 2.5 h . Then, the reactions were analyzed by western blot analysis using antibodies against AcK, DDB2 and SIRT6. The recombinant DDB2-GFP, SIRT6 WT or SIRT6 H133Y mutant proteins were also resolved on SDS-PAGE gels, followed by Coomassie Blue staining. The gel was stained with Coomassie reagent (ratio, methanol:acetic acid:Coomassie:H 2 O = 45:10:0.25:45) for 2.5 h, followed by washing with a destaining solution (ratio, methanol:acetic acid:H 2 O = 25:8:67). ( D ) The change in the acetylation level of exogenous DDB2 in response to UV irradiation was evaluated. HCA2-hTERT cells were transfected with a vector encoding DDB2-GFP and irradiated with or without UVC (20 J/m 2 ). Then, the cells were harvested at 5 min post UV irradiation. Cell lysates were immunoprecipitated with GFP-Trap, followed by western blot analysis with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP. ( E ) The changes in the acetylation levels of exogenous DDB2 in control and SIRT6-depleted cells in response to UV irradiation were evaluated. The control and SIRT6-depleted HCA2-hTERT cells were transfected with a vector encoding DDB2-GFP and irradiated with or without UVC (20 J/m 2 ). Then, the cells were harvested at 5 mins post UV irradiation. Cell lysates were immunoprecipitated with GFP-Trap, followed by western blot analysis with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP. ( F ) The acetylation levels of DDB2 fragments were assessed. HEK293 cells were transfected with vectors encoding the GFP-tagged DDB2 fragments, DDB2 fragment 1-GFP (1–100 aa) or DDB2 fragment 2-GFP (101–427 aa). At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against acetylated lysine, followed by western blot analysis. The arrow indicates the acetylated DDB2-F1-GFP. ( G ) The Coomassie blue-stained SDS-PAGE gel image shows that DDB2 immunoprecipitated from HEK293 cells with an anti-GFP antibody. HEK293 cells were transfected with a plasmid encoding DDB2-GFP. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against GFP, followed by SDS-PAGE. ( H ) A schematic representation of lysine residues in DDB2 fragment 1 (1–100 aa) is shown. ( I ) K35 and K77 are the two lysine residues that are acetylated in DDB2. DDB2-GFP WT and the indicated DDB2 mutants were transfected into HEK293 cells. GFP-tagged DDB2 was pulled down from cell lysates using an anti-acetylated lysine antibody and immunoblotted with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP WT or mutants. ( J ) The acetylation levels of DDB2-GFP WT and the 2KR mutant were measured. DDB2-GFP WT and DDB2-GFP 2KR were transfected into HEK293 cells. GFP-tagged DDB2 was pulled down from cell lysates using an anti-acetylated lysine antibody and immunoblotted with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP WT or mutant.
    Figure Legend Snippet: SIRT6 deacetylates DDB2 in response to UV irradiation. ( A ) The interaction between SIRT6 and DDB2 is enhanced in response to UVC irradiation. HEK293 cells with stable Flag-tagged SIRT6 integration were transfected with DDB2-GFP and treated with or without UVC (20 J/m 2 ). Then, cells were harvested at the indicated time points. Cell lysates were immunoprecipitated with an anti-Flag antibody, followed by western blot analysis with the indicated antibodies. IPed, immunoprecipitated. ( B ) DDB2 is a target of the deacetylase SIRT6. HEK293 cells were cotransfected with plasmids encoding DDB2-GFP and SIRT6-Flag. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against acetylated lysines (AcK), followed by western blot analysis with the indicated antibodies. ( C ) DDB2 is deacetylated by SIRT6 in vitro . A recombinant DDB2-GFP protein (5 μg) and a SIRT6 WT or H133Y mutant protein (5 μg) purified from HEK293 cells were incubated to allow the deacetylation reaction to occur in HDAC buffer (50 mM Tris-HCl pH 9.0, 4 mM MgCl 2 , 50 mM NaCl, 0.2 mM DTT and 1 mM NAD+) at 30°C for 2.5 h . Then, the reactions were analyzed by western blot analysis using antibodies against AcK, DDB2 and SIRT6. The recombinant DDB2-GFP, SIRT6 WT or SIRT6 H133Y mutant proteins were also resolved on SDS-PAGE gels, followed by Coomassie Blue staining. The gel was stained with Coomassie reagent (ratio, methanol:acetic acid:Coomassie:H 2 O = 45:10:0.25:45) for 2.5 h, followed by washing with a destaining solution (ratio, methanol:acetic acid:H 2 O = 25:8:67). ( D ) The change in the acetylation level of exogenous DDB2 in response to UV irradiation was evaluated. HCA2-hTERT cells were transfected with a vector encoding DDB2-GFP and irradiated with or without UVC (20 J/m 2 ). Then, the cells were harvested at 5 min post UV irradiation. Cell lysates were immunoprecipitated with GFP-Trap, followed by western blot analysis with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP. ( E ) The changes in the acetylation levels of exogenous DDB2 in control and SIRT6-depleted cells in response to UV irradiation were evaluated. The control and SIRT6-depleted HCA2-hTERT cells were transfected with a vector encoding DDB2-GFP and irradiated with or without UVC (20 J/m 2 ). Then, the cells were harvested at 5 mins post UV irradiation. Cell lysates were immunoprecipitated with GFP-Trap, followed by western blot analysis with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP. ( F ) The acetylation levels of DDB2 fragments were assessed. HEK293 cells were transfected with vectors encoding the GFP-tagged DDB2 fragments, DDB2 fragment 1-GFP (1–100 aa) or DDB2 fragment 2-GFP (101–427 aa). At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against acetylated lysine, followed by western blot analysis. The arrow indicates the acetylated DDB2-F1-GFP. ( G ) The Coomassie blue-stained SDS-PAGE gel image shows that DDB2 immunoprecipitated from HEK293 cells with an anti-GFP antibody. HEK293 cells were transfected with a plasmid encoding DDB2-GFP. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against GFP, followed by SDS-PAGE. ( H ) A schematic representation of lysine residues in DDB2 fragment 1 (1–100 aa) is shown. ( I ) K35 and K77 are the two lysine residues that are acetylated in DDB2. DDB2-GFP WT and the indicated DDB2 mutants were transfected into HEK293 cells. GFP-tagged DDB2 was pulled down from cell lysates using an anti-acetylated lysine antibody and immunoblotted with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP WT or mutants. ( J ) The acetylation levels of DDB2-GFP WT and the 2KR mutant were measured. DDB2-GFP WT and DDB2-GFP 2KR were transfected into HEK293 cells. GFP-tagged DDB2 was pulled down from cell lysates using an anti-acetylated lysine antibody and immunoblotted with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP WT or mutant.

    Techniques Used: Irradiation, Transfection, Immunoprecipitation, Western Blot, Histone Deacetylase Assay, In Vitro, Recombinant, Mutagenesis, Purification, Incubation, SDS Page, Staining, Plasmid Preparation

    Loss of SIRT6 leads to the retention of DDB2 on chromatin in response to UV irradiation. ( A ) Changes in DDB2 bound to chromatin in the absence of SIRT6 in response to UV irradiation were evaluated. Control and SIRT6-depleted HCA2-hTERT cells were irradiated with UVC (20 J/m 2 ), harvested at the indicated time points and subjected to cellular protein fractionation, followed by western blot analysis of DDB2 in the chromatin fractions. ( B ) Depleting SIRT6 abolished the increased ubiquitination of DDB2 upon UV irradiation. Control and SIRT6-depleted HEK293 cells were transfected with a plasmid encoding DDB2-Flag and treated with MG132 at 10 μM. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis with an antibody recognizing ubiquitin. ( C ) The ubiquitination level of the DDB2 2KR mutant was lower than that of DDB2 WT. HEK293 cells were transfected with a DDB2-Flag or DDB2-2KR-Flag plasmid. After 16 h, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis with an antibody recognizing ubiquitin. ( D ) DDB2 2KR partially abolished the interaction between DDB2 and p97. HEK293 cells were transfected with a pControl, DDB2-GFP or DDB2-2KR-GFP plasmid. After 16 h, the cells were irradiated with UVC or left untreated, and at 5 min post UV irradiation, the cells were harvested for immunoprecipitation with an antibody against GFP and immunoblotted with the indicated antibodies. IPed, immunoprecipitated. ( E ) SIRT6 greatly diminished the interaction between DDB2 and p97. Control and SIRT6-depleted HEK293 cells were transfected with a plasmid encoding DDB2-Flag. After 16 h, the cells were harvested for immunoprecipitation with an antibody against Flag and immunoblotted with the indicated antibodies. IPed, immunoprecipitated. ( F ) Immunoblot analysis of changes in XPC hyperubiquitination in the absence or presence of SIRT6 was performed. Control and SIRT6-depleted HCA2-hTERT cells were UVC irradiated (20 J/m 2 ) and harvested at the indicated time points, followed by western blot analysis.
    Figure Legend Snippet: Loss of SIRT6 leads to the retention of DDB2 on chromatin in response to UV irradiation. ( A ) Changes in DDB2 bound to chromatin in the absence of SIRT6 in response to UV irradiation were evaluated. Control and SIRT6-depleted HCA2-hTERT cells were irradiated with UVC (20 J/m 2 ), harvested at the indicated time points and subjected to cellular protein fractionation, followed by western blot analysis of DDB2 in the chromatin fractions. ( B ) Depleting SIRT6 abolished the increased ubiquitination of DDB2 upon UV irradiation. Control and SIRT6-depleted HEK293 cells were transfected with a plasmid encoding DDB2-Flag and treated with MG132 at 10 μM. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis with an antibody recognizing ubiquitin. ( C ) The ubiquitination level of the DDB2 2KR mutant was lower than that of DDB2 WT. HEK293 cells were transfected with a DDB2-Flag or DDB2-2KR-Flag plasmid. After 16 h, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis with an antibody recognizing ubiquitin. ( D ) DDB2 2KR partially abolished the interaction between DDB2 and p97. HEK293 cells were transfected with a pControl, DDB2-GFP or DDB2-2KR-GFP plasmid. After 16 h, the cells were irradiated with UVC or left untreated, and at 5 min post UV irradiation, the cells were harvested for immunoprecipitation with an antibody against GFP and immunoblotted with the indicated antibodies. IPed, immunoprecipitated. ( E ) SIRT6 greatly diminished the interaction between DDB2 and p97. Control and SIRT6-depleted HEK293 cells were transfected with a plasmid encoding DDB2-Flag. After 16 h, the cells were harvested for immunoprecipitation with an antibody against Flag and immunoblotted with the indicated antibodies. IPed, immunoprecipitated. ( F ) Immunoblot analysis of changes in XPC hyperubiquitination in the absence or presence of SIRT6 was performed. Control and SIRT6-depleted HCA2-hTERT cells were UVC irradiated (20 J/m 2 ) and harvested at the indicated time points, followed by western blot analysis.

    Techniques Used: Irradiation, Fractionation, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Mutagenesis

    Several mutations in SIRT6 in melanoma impair the repair of UV-induced DNA damage and result in a high incidence of mutation rates across the genome. ( A ) Lolliplot of the SIRT6 protein with the alterations present in melanoma samples indicated. ( B ) Locations of the alterations mapped to the SIRT6 crystal structure (PDB: 3ZG6). ( C ) Number of nonsynonymous mutations in melanoma samples with SIRT6 mutations. ( D ) Several SIRT6 mutants lost the ability to enhance NER efficiency. Error bars represent the s.d. *** P < 0.001, ** P < 0.01, n.s., not significant. ( E ) The SIRT6 P27S and H50Y mutants partially lost their deacetylase activity. HEK293 cells were cotransfected with plasmids encoding DDB2-GFP and SIRT6 WT or mutants. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against acetylated lysine, followed by western blot analysis with the indicated antibodies. ( F ) SIRT6 G134W has a high turnover rate. HCA2-hTERT cells were harvested for protein extraction at 3 and 24 h post transfection with a control vector, SIRT6 WT or G134W mutant, followed by western blot analysis of SIRT6 expression.
    Figure Legend Snippet: Several mutations in SIRT6 in melanoma impair the repair of UV-induced DNA damage and result in a high incidence of mutation rates across the genome. ( A ) Lolliplot of the SIRT6 protein with the alterations present in melanoma samples indicated. ( B ) Locations of the alterations mapped to the SIRT6 crystal structure (PDB: 3ZG6). ( C ) Number of nonsynonymous mutations in melanoma samples with SIRT6 mutations. ( D ) Several SIRT6 mutants lost the ability to enhance NER efficiency. Error bars represent the s.d. *** P < 0.001, ** P < 0.01, n.s., not significant. ( E ) The SIRT6 P27S and H50Y mutants partially lost their deacetylase activity. HEK293 cells were cotransfected with plasmids encoding DDB2-GFP and SIRT6 WT or mutants. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against acetylated lysine, followed by western blot analysis with the indicated antibodies. ( F ) SIRT6 G134W has a high turnover rate. HCA2-hTERT cells were harvested for protein extraction at 3 and 24 h post transfection with a control vector, SIRT6 WT or G134W mutant, followed by western blot analysis of SIRT6 expression.

    Techniques Used: Mutagenesis, Histone Deacetylase Assay, Activity Assay, Transfection, Immunoprecipitation, Western Blot, Protein Extraction, Plasmid Preparation, Expressing

    rabbit anti dna binding protein 2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti dna binding protein 2
    p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; <t>DDB2,</t> DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.
    Rabbit Anti Dna Binding Protein 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells"

    Article Title: Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00160.2018

    p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; DDB2, DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.
    Figure Legend Snippet: p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; DDB2, DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.

    Techniques Used: Immunolabeling, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Binding Assay

    p53 transactivation domain (TAD) deletion impairs DNA damage repair in human induced pluripotent stem cells (hiPSCs). A and B, left: Western blots of Rad50, Mre11, phosphorylated checkpoint kinase 2 (p-Chk2) at Thr68, and total Chk2 (A) as well as xeroderma pigmentosum group D (XPD), xeroderma pigmentosum group A (XPA), DNA-binding protein 2 (DDB2), and DNA polymerase H (DNA Pol η) (B) in wild-type (WT) and p53-TAD knockout (KO) hiPSCs with and without doxorubicin (Doxo) treatment. Western blot quantifications are shown on the right. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. the respective nontreated cells; †P < 0.05 vs. WT; ‡P < 0.05 vs. WT + Doxo. C: p53-TAD hiPSCs were immunolabeled for γH2A.X (green; left) and counterstained with DAPI (blue; right). D: schematic of DNA damage repair methodology. E: nucleoids in WT hiPSCs and p53-TAD KO hiPSCs treated with Doxo (top) and after recovery (bottom) were stained with Vista green dye (green). Comets were apparent with Doxo and after recovery of p53-TAD KO hiPSCs, whereas intact DNA was noted in WT hiPSCs after recovery. F: tail moment of WT hiPSCs and p53-TAD KO hiPSCs nuclei at baseline (control), after Doxo, and after recovery. a.u., arbitrary units. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. control; **P < 0.05 vs. Doxo. G: representative micrograph of hiPSCs immunolabeled for γH2A.X (green; left) and nuclei stained with DAPI (blue; right). H: fraction of WT p53 and p53-TAD KO hiPSCs positive for γH2A.X. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. WT.
    Figure Legend Snippet: p53 transactivation domain (TAD) deletion impairs DNA damage repair in human induced pluripotent stem cells (hiPSCs). A and B, left: Western blots of Rad50, Mre11, phosphorylated checkpoint kinase 2 (p-Chk2) at Thr68, and total Chk2 (A) as well as xeroderma pigmentosum group D (XPD), xeroderma pigmentosum group A (XPA), DNA-binding protein 2 (DDB2), and DNA polymerase H (DNA Pol η) (B) in wild-type (WT) and p53-TAD knockout (KO) hiPSCs with and without doxorubicin (Doxo) treatment. Western blot quantifications are shown on the right. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. the respective nontreated cells; †P < 0.05 vs. WT; ‡P < 0.05 vs. WT + Doxo. C: p53-TAD hiPSCs were immunolabeled for γH2A.X (green; left) and counterstained with DAPI (blue; right). D: schematic of DNA damage repair methodology. E: nucleoids in WT hiPSCs and p53-TAD KO hiPSCs treated with Doxo (top) and after recovery (bottom) were stained with Vista green dye (green). Comets were apparent with Doxo and after recovery of p53-TAD KO hiPSCs, whereas intact DNA was noted in WT hiPSCs after recovery. F: tail moment of WT hiPSCs and p53-TAD KO hiPSCs nuclei at baseline (control), after Doxo, and after recovery. a.u., arbitrary units. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. control; **P < 0.05 vs. Doxo. G: representative micrograph of hiPSCs immunolabeled for γH2A.X (green; left) and nuclei stained with DAPI (blue; right). H: fraction of WT p53 and p53-TAD KO hiPSCs positive for γH2A.X. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. WT.

    Techniques Used: Western Blot, Binding Assay, Knock-Out, Immunolabeling, Staining

    ddb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ddb2
    (A) SW480 cells and HT-29 cells were transient transfected with siRNAs (siControl or siDDB2). Total RNA was analyzed using qRT-PCR for the mRNA level of Rnf43 N=3, Error bars indicate SD. (B) Total levels of <t>DDB2,</t> RNF43 and α-Tubulin (Loading Control) proteins were analyzed by Western Blotting. (C and D) The indicated cell lines were infected with adenovirus expressing either LacZ or T7 tagged DDB2 (T7-DDB2). qRT-PCR (C) was performed to analyze relative mRNA level of Rnf43expression (Normalized by gapdh). N=3, Error bars indicate SD. Western blotting (D) was performed to analyze the protein levels of DDB2, RNF43 and α-Tubulin. (E and F) Immunohistochemical staining of RNF43 and DDB2 in adjacent normal, adjacent foci of hyperplasia, dysplasia and low-grade adenocarcinoma samples were quantified as positivity (=Number of Positive/Number of Total). Positivity of DDB2 (E) and RNF43 (F) were plotted. N of Adjacent normal = 47, N of Adjacent foci of hyperplasia= 42, N of Dysplasia = 18, N of Low-grade adenocarcinoma = 47. **** P < 0.0001 (G) Representative images of DDB2 expression and RNF43 expression from same patient. Scale bar =50 µm.
    Ddb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "DDB2 is a Novel Regulator of Wnt-Signaling in Colon Cancer"

    Article Title: DDB2 is a Novel Regulator of Wnt-Signaling in Colon Cancer

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-17-1570

    (A) SW480 cells and HT-29 cells were transient transfected with siRNAs (siControl or siDDB2). Total RNA was analyzed using qRT-PCR for the mRNA level of Rnf43 N=3, Error bars indicate SD. (B) Total levels of DDB2, RNF43 and α-Tubulin (Loading Control) proteins were analyzed by Western Blotting. (C and D) The indicated cell lines were infected with adenovirus expressing either LacZ or T7 tagged DDB2 (T7-DDB2). qRT-PCR (C) was performed to analyze relative mRNA level of Rnf43expression (Normalized by gapdh). N=3, Error bars indicate SD. Western blotting (D) was performed to analyze the protein levels of DDB2, RNF43 and α-Tubulin. (E and F) Immunohistochemical staining of RNF43 and DDB2 in adjacent normal, adjacent foci of hyperplasia, dysplasia and low-grade adenocarcinoma samples were quantified as positivity (=Number of Positive/Number of Total). Positivity of DDB2 (E) and RNF43 (F) were plotted. N of Adjacent normal = 47, N of Adjacent foci of hyperplasia= 42, N of Dysplasia = 18, N of Low-grade adenocarcinoma = 47. **** P < 0.0001 (G) Representative images of DDB2 expression and RNF43 expression from same patient. Scale bar =50 µm.
    Figure Legend Snippet: (A) SW480 cells and HT-29 cells were transient transfected with siRNAs (siControl or siDDB2). Total RNA was analyzed using qRT-PCR for the mRNA level of Rnf43 N=3, Error bars indicate SD. (B) Total levels of DDB2, RNF43 and α-Tubulin (Loading Control) proteins were analyzed by Western Blotting. (C and D) The indicated cell lines were infected with adenovirus expressing either LacZ or T7 tagged DDB2 (T7-DDB2). qRT-PCR (C) was performed to analyze relative mRNA level of Rnf43expression (Normalized by gapdh). N=3, Error bars indicate SD. Western blotting (D) was performed to analyze the protein levels of DDB2, RNF43 and α-Tubulin. (E and F) Immunohistochemical staining of RNF43 and DDB2 in adjacent normal, adjacent foci of hyperplasia, dysplasia and low-grade adenocarcinoma samples were quantified as positivity (=Number of Positive/Number of Total). Positivity of DDB2 (E) and RNF43 (F) were plotted. N of Adjacent normal = 47, N of Adjacent foci of hyperplasia= 42, N of Dysplasia = 18, N of Low-grade adenocarcinoma = 47. **** P < 0.0001 (G) Representative images of DDB2 expression and RNF43 expression from same patient. Scale bar =50 µm.

    Techniques Used: Transfection, Quantitative RT-PCR, Western Blot, Infection, Expressing, Immunohistochemical staining, Staining

    (A) The schematic representation of the human Rnf43upstream regulatory region. The thick truncated lines mark the regions covered by primer sets of interest. TSS: Transcription start site. (B) Chromatin-IP (ChIP) assays for binding of DDB2 with regulatory region upstream of the Rnf43gene. The ChIP assay was conducted to analyze the local enrichment of DDB2 across the upstream regulatory region and part of 5’ UTR of Rnf43gene in HCT 116 cells. The relative fold enrichment was quantified by normalization to input. And the fold enrichment of IgG is set as 1 for all primer sets. N = 5, Error bars indicate SD. **: P <0.01. (C) The schematic representation of the human Rnf43upstream regulatory region. The thick truncated lines mark the P2 and P3 regions. The white rectangle between P2 and P3 regions indicates the putative DDB2 binding element ‘TCCCCTAA’. Dash lines indicate location of the sequences targeted by sgRNAs used in CRISPR/Cas9 genome editing. The orange arrowheads indicate the precise region where the Cas9 Nuclease cuts the genomic sequence. (D and E) ChIP assay was performed to analyze the local enrichment of DDB2 in HT-29 Parental cells (D) and Short Del cells (E). The relative fold enrichment was quantified by normalization to input. And the fold enrichment of IgG is set as 1 for all primer sets. (F–H) Parental HT-29 cells, Short Del HT-29 cells and two clones of Long Del HT-29 cells were treated with DMSO or CHIR99021 (6.7µM, 24 hrs). The mRNA levels of DDB2 (F), Rnf43 (G) and Axin2 (H) were analyzed using qRT-PCR (Normalized by 18S rRNA). N = 3, Error bars indicate SD. **: P <0.01. ***: P <0.001
    Figure Legend Snippet: (A) The schematic representation of the human Rnf43upstream regulatory region. The thick truncated lines mark the regions covered by primer sets of interest. TSS: Transcription start site. (B) Chromatin-IP (ChIP) assays for binding of DDB2 with regulatory region upstream of the Rnf43gene. The ChIP assay was conducted to analyze the local enrichment of DDB2 across the upstream regulatory region and part of 5’ UTR of Rnf43gene in HCT 116 cells. The relative fold enrichment was quantified by normalization to input. And the fold enrichment of IgG is set as 1 for all primer sets. N = 5, Error bars indicate SD. **: P <0.01. (C) The schematic representation of the human Rnf43upstream regulatory region. The thick truncated lines mark the P2 and P3 regions. The white rectangle between P2 and P3 regions indicates the putative DDB2 binding element ‘TCCCCTAA’. Dash lines indicate location of the sequences targeted by sgRNAs used in CRISPR/Cas9 genome editing. The orange arrowheads indicate the precise region where the Cas9 Nuclease cuts the genomic sequence. (D and E) ChIP assay was performed to analyze the local enrichment of DDB2 in HT-29 Parental cells (D) and Short Del cells (E). The relative fold enrichment was quantified by normalization to input. And the fold enrichment of IgG is set as 1 for all primer sets. (F–H) Parental HT-29 cells, Short Del HT-29 cells and two clones of Long Del HT-29 cells were treated with DMSO or CHIR99021 (6.7µM, 24 hrs). The mRNA levels of DDB2 (F), Rnf43 (G) and Axin2 (H) were analyzed using qRT-PCR (Normalized by 18S rRNA). N = 3, Error bars indicate SD. **: P <0.01. ***: P <0.001

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, CRISPR, Sequencing, Clone Assay, Quantitative RT-PCR

    (A) Nuclear extracts of HCT 116 cells expressing empty vector or Flag-HA-DDB2 were immunoprecipitated by ANTI-FLAG M2 affinity gel and analyzed by Western Blotting using DDB2 and total β-catenin antibodies. IP: Immunoprecipitation; IB: Immunoblotting. (B and C) ChIP assay was performed to analyze the local enrichment of DDB2 and β-catenin across the upstream regulatory region and part of 5’ UTR of Rnf43gene in HCT 116 cells expressing either shControl (B) or shDDB2 (C). The relative fold enrichment was quantified by normalization to input. And the fold enrichment of IgG is set as 1 for all primer sets. A primer set which amplifies the TCF/ β-catenin binding site on Axin2 intron1 (Cell Signaling) is used as positive control for β-catenin immunoprecipitation. (D and E) ChIP assay was performed to analyze the local enrichment of β-catenin from P1 to P5 regions of Rnf43gene in HT-29 Parental (D) and Short Del (E) cells. The relative fold enrichment was quantified by normalization to input. And the fold enrichment of IgG is set as 1 for all primer sets. A primer set which amplifies the TCF/ β-catenin binding site on c-Myc promoter is used as positive control. N = 3, Error bars indicate SD. **: P <0.01. ***: P <0.001
    Figure Legend Snippet: (A) Nuclear extracts of HCT 116 cells expressing empty vector or Flag-HA-DDB2 were immunoprecipitated by ANTI-FLAG M2 affinity gel and analyzed by Western Blotting using DDB2 and total β-catenin antibodies. IP: Immunoprecipitation; IB: Immunoblotting. (B and C) ChIP assay was performed to analyze the local enrichment of DDB2 and β-catenin across the upstream regulatory region and part of 5’ UTR of Rnf43gene in HCT 116 cells expressing either shControl (B) or shDDB2 (C). The relative fold enrichment was quantified by normalization to input. And the fold enrichment of IgG is set as 1 for all primer sets. A primer set which amplifies the TCF/ β-catenin binding site on Axin2 intron1 (Cell Signaling) is used as positive control for β-catenin immunoprecipitation. (D and E) ChIP assay was performed to analyze the local enrichment of β-catenin from P1 to P5 regions of Rnf43gene in HT-29 Parental (D) and Short Del (E) cells. The relative fold enrichment was quantified by normalization to input. And the fold enrichment of IgG is set as 1 for all primer sets. A primer set which amplifies the TCF/ β-catenin binding site on c-Myc promoter is used as positive control. N = 3, Error bars indicate SD. **: P <0.01. ***: P <0.001

    Techniques Used: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Binding Assay, Positive Control

    (A) Nuclear extracts of HCT 116 cells expressing empty vector or Flag-HA-DDB2 were immunoprecipitated by ANTI-FLAG M2 affinity gel and analyzed by Western Blotting using EZH2, EED and SUZ12 antibodies. IP: Immunoprecipitation; IB: Immunoblotting. (B) ChIP assay was conducted to analyze the local enrichment of DDB2 and EZH2 at P2 region in HCT 116 cells expressing shControl or shDDB2. The relative fold enrichment was quantified by normalization to input, and the fold enrichment of IgG is set as 1. N = 3, Error bars indicate SD. ***: P <0.001. (C) Nuclear extracts of HCT 116 cells transfected with siRNA-Control or siRNA-EZH2 were immunoprecipitated by normal Rabbit IgG or DDB2 antibodies. The results were analyzed by Western Blotting using DDB2, EZH2 and β-catenin antibodies. IP: Immunoprecipitation; IB: Immunoblotting. (D–F) EZH2 mediates DDB2- β-catenin interaction on P2 and P3 regions. The ChIP assays were conducted to analyze the enrichment of DDB2, EZH2 and β-catenin at P1(D), P2(E) and P3(F) regions in HCT 116 cells transfected with siRNA-Control or siRNA-EZH2. The relative fold enrichment was quantified by normalization to input, and the fold enrichment of IgG is set as 1. N = 3, Error bars indicate SD. *: P <0.05,**: P <0.01,***: P <0.001
    Figure Legend Snippet: (A) Nuclear extracts of HCT 116 cells expressing empty vector or Flag-HA-DDB2 were immunoprecipitated by ANTI-FLAG M2 affinity gel and analyzed by Western Blotting using EZH2, EED and SUZ12 antibodies. IP: Immunoprecipitation; IB: Immunoblotting. (B) ChIP assay was conducted to analyze the local enrichment of DDB2 and EZH2 at P2 region in HCT 116 cells expressing shControl or shDDB2. The relative fold enrichment was quantified by normalization to input, and the fold enrichment of IgG is set as 1. N = 3, Error bars indicate SD. ***: P <0.001. (C) Nuclear extracts of HCT 116 cells transfected with siRNA-Control or siRNA-EZH2 were immunoprecipitated by normal Rabbit IgG or DDB2 antibodies. The results were analyzed by Western Blotting using DDB2, EZH2 and β-catenin antibodies. IP: Immunoprecipitation; IB: Immunoblotting. (D–F) EZH2 mediates DDB2- β-catenin interaction on P2 and P3 regions. The ChIP assays were conducted to analyze the enrichment of DDB2, EZH2 and β-catenin at P1(D), P2(E) and P3(F) regions in HCT 116 cells transfected with siRNA-Control or siRNA-EZH2. The relative fold enrichment was quantified by normalization to input, and the fold enrichment of IgG is set as 1. N = 3, Error bars indicate SD. *: P <0.05,**: P <0.01,***: P <0.001

    Techniques Used: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Transfection

    (A) The expression of dominant negative TCF4 decreased Rnf43expression. HCT 116 cells stably expressing empty vector or dominant-negative TCF4 (dn-TCF4) were treated with DMSO or CHIR99021 (3.3µM, 16hrs). Relative mRNA levels of Rnf43and Axin2 were analyzed by qRT-PCR. N = 3, Error bars indicate SD. (B) Expression of dominant negative TCF4 reduced EZH2 and β-catenin interaction with TCF4 binding regions. The ChIP assay was performed in HCT 116 cells stably expressing empty vector or dn-TCF4. The enrichment of DDB2, EZH2 and β-catenin was analyzed at P2, P3 as well as at the TCF4 binding sites in the intron regions of Rnf43gene. The relative fold enrichment was quantified by normalization to input, and the fold enrichment of IgG is set as 1. N = 5, Error bars indicate SD. *: P <0.05,**: P <0.01. (C) A schematic diagram showing the location of primer and probe sets designed for the Chromatin Conformation Capture (3C) assay. The white rectangle indicates the putative DDB2 binding element at upstream regulatory region in the Rnf43gene. The black bars mark two binding sites of TCF4 in intron2 of the Rnf43gene. The black truncated lines across the Rnf43gene indicate the sites that are recognized by BglII restriction endonuclease. The arrows mark the primers for each of the primer sets. Four sets of primers are designed to detect physical interactions between restriction fragments. (D and E) Expression of DDB2-shRNA (D) or expression of a dominant negative mutant of TCF4 (E) decreases relative crosslinking frequency at Set2. The 3C assays were conducted in HCT 116 cells expressing shControl vs. shDDB2 (D); or control vector vs. dn-TCF expression vector (E). Four sets of primers and probes were analyzed using Taqman qPCR. A serial dilution of the control template was used for standard curve; and the relative crosslinking frequencies were normalized by gapdh loading control. N=4, Error bars indicate SD.
    Figure Legend Snippet: (A) The expression of dominant negative TCF4 decreased Rnf43expression. HCT 116 cells stably expressing empty vector or dominant-negative TCF4 (dn-TCF4) were treated with DMSO or CHIR99021 (3.3µM, 16hrs). Relative mRNA levels of Rnf43and Axin2 were analyzed by qRT-PCR. N = 3, Error bars indicate SD. (B) Expression of dominant negative TCF4 reduced EZH2 and β-catenin interaction with TCF4 binding regions. The ChIP assay was performed in HCT 116 cells stably expressing empty vector or dn-TCF4. The enrichment of DDB2, EZH2 and β-catenin was analyzed at P2, P3 as well as at the TCF4 binding sites in the intron regions of Rnf43gene. The relative fold enrichment was quantified by normalization to input, and the fold enrichment of IgG is set as 1. N = 5, Error bars indicate SD. *: P <0.05,**: P <0.01. (C) A schematic diagram showing the location of primer and probe sets designed for the Chromatin Conformation Capture (3C) assay. The white rectangle indicates the putative DDB2 binding element at upstream regulatory region in the Rnf43gene. The black bars mark two binding sites of TCF4 in intron2 of the Rnf43gene. The black truncated lines across the Rnf43gene indicate the sites that are recognized by BglII restriction endonuclease. The arrows mark the primers for each of the primer sets. Four sets of primers are designed to detect physical interactions between restriction fragments. (D and E) Expression of DDB2-shRNA (D) or expression of a dominant negative mutant of TCF4 (E) decreases relative crosslinking frequency at Set2. The 3C assays were conducted in HCT 116 cells expressing shControl vs. shDDB2 (D); or control vector vs. dn-TCF expression vector (E). Four sets of primers and probes were analyzed using Taqman qPCR. A serial dilution of the control template was used for standard curve; and the relative crosslinking frequencies were normalized by gapdh loading control. N=4, Error bars indicate SD.

    Techniques Used: Expressing, Dominant Negative Mutation, Stable Transfection, Plasmid Preparation, Quantitative RT-PCR, Binding Assay, shRNA, Serial Dilution

    (A) DDB2 deficiency leads to inhibition of FZD5 ubiquitination. HT-29 cells were transfected with V5–FZD5, His-ubiquitin and siRNA-Control or siRNA-DDB2, as indicated. His-Ubiquitinated proteins were collected by nickel-nitrilotriacetic acid–agarose beads and the presence of ubiquitinated V5-FZD5 is detected by V5 antibody. (B) Lack of DDB2 bound region in Rnf43gene results in inhibition of FZD5 ubiquitination. HT-29 Parental, Short Del and Long Del cells were transfected with V5–FZD5 and His-ubiquitin. And the assay was performed as described above. IP, Immunopull-down; IB: Immunoblotting; Ub, ubiquitin. Arrows indicate mature, glycosylated V5-conjugated FZD5. (C) HT-29 cells transiently expressing siControl or siDDB2 were treated with vehicle or human recombinant Wnt3A protein (300ng/ml, 24hrs). Relative mRNA levels of DDB2, Rnf43, Axin2 and c-Myc were analyzed using qRT-PCR (Normalized by 18S rRNA). N=3. All error bars indicate SD. (D) Lack of DDB2 binding region in Rnf43gene increases the expression of Wnt/β-catenin pathway target genes upon Wnt3a treatment. HT-29 Parental cells, Short Del cells and Long Del cells were treated with vehicle or human recombinant Wnt3A protein (300ng/ml, 24hrs). Relative mRNA levels of DDB2, Rnf43, Axin2 and c-Myc were analyzed using qRT-PCR (Normalized by 18S rRNA). N=3. All error bars indicate SD. *: P <0.05, **: P <0.01, ***: P <0.001
    Figure Legend Snippet: (A) DDB2 deficiency leads to inhibition of FZD5 ubiquitination. HT-29 cells were transfected with V5–FZD5, His-ubiquitin and siRNA-Control or siRNA-DDB2, as indicated. His-Ubiquitinated proteins were collected by nickel-nitrilotriacetic acid–agarose beads and the presence of ubiquitinated V5-FZD5 is detected by V5 antibody. (B) Lack of DDB2 bound region in Rnf43gene results in inhibition of FZD5 ubiquitination. HT-29 Parental, Short Del and Long Del cells were transfected with V5–FZD5 and His-ubiquitin. And the assay was performed as described above. IP, Immunopull-down; IB: Immunoblotting; Ub, ubiquitin. Arrows indicate mature, glycosylated V5-conjugated FZD5. (C) HT-29 cells transiently expressing siControl or siDDB2 were treated with vehicle or human recombinant Wnt3A protein (300ng/ml, 24hrs). Relative mRNA levels of DDB2, Rnf43, Axin2 and c-Myc were analyzed using qRT-PCR (Normalized by 18S rRNA). N=3. All error bars indicate SD. (D) Lack of DDB2 binding region in Rnf43gene increases the expression of Wnt/β-catenin pathway target genes upon Wnt3a treatment. HT-29 Parental cells, Short Del cells and Long Del cells were treated with vehicle or human recombinant Wnt3A protein (300ng/ml, 24hrs). Relative mRNA levels of DDB2, Rnf43, Axin2 and c-Myc were analyzed using qRT-PCR (Normalized by 18S rRNA). N=3. All error bars indicate SD. *: P <0.05, **: P <0.01, ***: P <0.001

    Techniques Used: Inhibition, Transfection, Western Blot, Expressing, Recombinant, Quantitative RT-PCR, Binding Assay

    (A) Schematic of the AOM/DSS protocol. Mice were injected with AOM one week prior to beginning the 7-day treatment of DSS. AOM/DSS treated mice were sacrificed 15 weeks after AOM injection. (B) Representative images of mouse colorectum. At 15 weeks, male DDB2+/+ and DDB2−/− mice were sacrificed and the entire colorectal tissues were excised and whole mounts were examined from proximal to distal ends using light microscopy. Increased numbers of nodular and polyploid colonic tumors were observed in the colorectum of the DDB2−/− mice. (C) DDB2 −/− mice developed greater number of tumor nodules than wild-type mice. Quantification of total tumor nodules and the tumors larger than 2-mm diameter observed in 12 DDB2 +/+ mice and 14 DDB2−/− mice. (D) Colorectal tumors from DDB2−/− mice showed significantly decreased level of RNF43 expression and up-regulated level of LRP5/6. Immunohistochemical staining for DDB2, RNF43 and LRP5/6 were performed on the colorectal tissue sections from DDB2+/+ and DDB2−/− mice and images were taken under 40x objective. (Scale bar=50 µm). (E)Western blotting shows the knockout of DDB2 in colon tumor samples. (F) Quantification of the percentage of RNF43 positive and LRP5/6 positive cells. N=15. (G) Upregulated expression of Wnt target genes in DDB2−/− tumors. RNA was extracted from colorectal tumors isolated from DDB2+/+ mice and DDB2−/− mice, and the relative mRNA levels of Rnf43, Axin2 and Cdx1 were examined by qPCR. N=3. All error bars indicate SD. *: P <0.05, **: P <0.01, ***: P <0.001
    Figure Legend Snippet: (A) Schematic of the AOM/DSS protocol. Mice were injected with AOM one week prior to beginning the 7-day treatment of DSS. AOM/DSS treated mice were sacrificed 15 weeks after AOM injection. (B) Representative images of mouse colorectum. At 15 weeks, male DDB2+/+ and DDB2−/− mice were sacrificed and the entire colorectal tissues were excised and whole mounts were examined from proximal to distal ends using light microscopy. Increased numbers of nodular and polyploid colonic tumors were observed in the colorectum of the DDB2−/− mice. (C) DDB2 −/− mice developed greater number of tumor nodules than wild-type mice. Quantification of total tumor nodules and the tumors larger than 2-mm diameter observed in 12 DDB2 +/+ mice and 14 DDB2−/− mice. (D) Colorectal tumors from DDB2−/− mice showed significantly decreased level of RNF43 expression and up-regulated level of LRP5/6. Immunohistochemical staining for DDB2, RNF43 and LRP5/6 were performed on the colorectal tissue sections from DDB2+/+ and DDB2−/− mice and images were taken under 40x objective. (Scale bar=50 µm). (E)Western blotting shows the knockout of DDB2 in colon tumor samples. (F) Quantification of the percentage of RNF43 positive and LRP5/6 positive cells. N=15. (G) Upregulated expression of Wnt target genes in DDB2−/− tumors. RNA was extracted from colorectal tumors isolated from DDB2+/+ mice and DDB2−/− mice, and the relative mRNA levels of Rnf43, Axin2 and Cdx1 were examined by qPCR. N=3. All error bars indicate SD. *: P <0.05, **: P <0.01, ***: P <0.001

    Techniques Used: Injection, Light Microscopy, Expressing, Immunohistochemical staining, Staining, Western Blot, Knock-Out, Isolation

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    USP44 deubiquitinates DDB2 following UVC exposure. (A,B) MEFs of the indicated genotype were exposed to 30 J/m 2 UVC and samples were collected for immunoblot at the indicated times. The doublet bands were consistently observed in these mouse cells, and were both quantitated to represent DDB2 using imageJ. The graph represents the mean ± SEM for three independent MEF lines. “*” denotes p < 0.05. (C) MEFs were transduced with the indicated constructs and subjected to immunoprecipitation for the FLAG epitope and probed as indicated. (D) Purified CRL-DDB2 was auto-ubiquitinated by the addition of UbE1, UBCH5a, and ubiquitin, and subsequently incubated with either wild type or catalytic mutant (C281A) immunopurified USP44 as indicated. Rxn = reaction.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: USP44 Stabilizes DDB2 to Facilitate Nucleotide Excision Repair and Prevent Tumors

    doi: 10.3389/fcell.2021.663411

    Figure Lengend Snippet: USP44 deubiquitinates DDB2 following UVC exposure. (A,B) MEFs of the indicated genotype were exposed to 30 J/m 2 UVC and samples were collected for immunoblot at the indicated times. The doublet bands were consistently observed in these mouse cells, and were both quantitated to represent DDB2 using imageJ. The graph represents the mean ± SEM for three independent MEF lines. “*” denotes p < 0.05. (C) MEFs were transduced with the indicated constructs and subjected to immunoprecipitation for the FLAG epitope and probed as indicated. (D) Purified CRL-DDB2 was auto-ubiquitinated by the addition of UbE1, UBCH5a, and ubiquitin, and subsequently incubated with either wild type or catalytic mutant (C281A) immunopurified USP44 as indicated. Rxn = reaction.

    Article Snippet: Antibodies used in this study include anti-DDB2 (#5416), FLAG (DYKDDDDK, #2368), histone H2B (#5546), α-actin (#4970) (Cell Signaling Inc. Danvers, MA, United States), HA (3F10, Millipore-Sigma, St. Louis, MO, United States), V5 (Bethyl Laboratories, Montgomery, TX, United States), CPD (clone TDM-2; CosmoBio) XPC (graciously supplied by Jeff Salisbury) ( ).

    Techniques: Western Blot, Transduction, Construct, Immunoprecipitation, FLAG-tag, Purification, Incubation, Mutagenesis

    Incomplete CPD repair in Usp44 null cells associated with inadequate DDB2 recruitment to sites of damage. (A,B) VH10 cells expressing DDB2-GFP were transfected with control or USP44 targeting siRNA and then locally irradiated. The DDB-GFP fluorescence was monitored over time using live-cell confocal imaging and quantified to pre-damage intensity set at 100. The graph represents the mean ± SEM for 31–36 cells per condition. (C) MEFs of the indicated genotypes were transduced with wild-type, catalytic mutant (USP44 C I ; C281A), centrin-binding deficient (USP44 C BM ; W162A) USP44, or DDB2 as indicated. The cells were exposed to UVC (10 J/m 2 ) and CPD levels were monitored at the indicated times. The graph represents the means of three independent experiments for each condition.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: USP44 Stabilizes DDB2 to Facilitate Nucleotide Excision Repair and Prevent Tumors

    doi: 10.3389/fcell.2021.663411

    Figure Lengend Snippet: Incomplete CPD repair in Usp44 null cells associated with inadequate DDB2 recruitment to sites of damage. (A,B) VH10 cells expressing DDB2-GFP were transfected with control or USP44 targeting siRNA and then locally irradiated. The DDB-GFP fluorescence was monitored over time using live-cell confocal imaging and quantified to pre-damage intensity set at 100. The graph represents the mean ± SEM for 31–36 cells per condition. (C) MEFs of the indicated genotypes were transduced with wild-type, catalytic mutant (USP44 C I ; C281A), centrin-binding deficient (USP44 C BM ; W162A) USP44, or DDB2 as indicated. The cells were exposed to UVC (10 J/m 2 ) and CPD levels were monitored at the indicated times. The graph represents the means of three independent experiments for each condition.

    Article Snippet: Antibodies used in this study include anti-DDB2 (#5416), FLAG (DYKDDDDK, #2368), histone H2B (#5546), α-actin (#4970) (Cell Signaling Inc. Danvers, MA, United States), HA (3F10, Millipore-Sigma, St. Louis, MO, United States), V5 (Bethyl Laboratories, Montgomery, TX, United States), CPD (clone TDM-2; CosmoBio) XPC (graciously supplied by Jeff Salisbury) ( ).

    Techniques: Expressing, Transfection, Irradiation, Fluorescence, Imaging, Transduction, Mutagenesis, Binding Assay

    p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; DDB2, DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells

    doi: 10.1152/ajpheart.00160.2018

    Figure Lengend Snippet: p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; DDB2, DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.

    Article Snippet: The following antibodies were used: NH 2 -terminal-specific rabbit monoclonal anti-p53 (Cell Signaling Technology, Danvers, MA), rabbit COOH-terminal anti-p53 (Sigma-Aldrich), rabbit monoclonal anti-checkpoint kinase 2 (Chk2; Thr 68 , Cell Signaling Technology), rabbit monoclonal anti-Chk2 (Cell Signaling Technology), rabbit monoclonal anti-Mre11 (Cell Signaling Technology), rabbit anti-Rad50 (Cell Signaling Technology), rabbit anti-xeroderma pigmentosum group D (XPD; D3Z6I, Cell Signaling Technology), rabbit anti-xeroderma pigmentosum group A (XPA; D9U5U, Cell Signaling Technology), rabbit anti-DNA polymerase H (DNA Pol η; E1I7T, Cell Signaling Technology), and rabbit anti-DNA-binding protein 2 (DDB2; D4C4, Cell Signaling Technology).

    Techniques: Immunolabeling, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Binding Assay

    p53 transactivation domain (TAD) deletion impairs DNA damage repair in human induced pluripotent stem cells (hiPSCs). A and B, left: Western blots of Rad50, Mre11, phosphorylated checkpoint kinase 2 (p-Chk2) at Thr68, and total Chk2 (A) as well as xeroderma pigmentosum group D (XPD), xeroderma pigmentosum group A (XPA), DNA-binding protein 2 (DDB2), and DNA polymerase H (DNA Pol η) (B) in wild-type (WT) and p53-TAD knockout (KO) hiPSCs with and without doxorubicin (Doxo) treatment. Western blot quantifications are shown on the right. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. the respective nontreated cells; †P < 0.05 vs. WT; ‡P < 0.05 vs. WT + Doxo. C: p53-TAD hiPSCs were immunolabeled for γH2A.X (green; left) and counterstained with DAPI (blue; right). D: schematic of DNA damage repair methodology. E: nucleoids in WT hiPSCs and p53-TAD KO hiPSCs treated with Doxo (top) and after recovery (bottom) were stained with Vista green dye (green). Comets were apparent with Doxo and after recovery of p53-TAD KO hiPSCs, whereas intact DNA was noted in WT hiPSCs after recovery. F: tail moment of WT hiPSCs and p53-TAD KO hiPSCs nuclei at baseline (control), after Doxo, and after recovery. a.u., arbitrary units. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. control; **P < 0.05 vs. Doxo. G: representative micrograph of hiPSCs immunolabeled for γH2A.X (green; left) and nuclei stained with DAPI (blue; right). H: fraction of WT p53 and p53-TAD KO hiPSCs positive for γH2A.X. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. WT.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells

    doi: 10.1152/ajpheart.00160.2018

    Figure Lengend Snippet: p53 transactivation domain (TAD) deletion impairs DNA damage repair in human induced pluripotent stem cells (hiPSCs). A and B, left: Western blots of Rad50, Mre11, phosphorylated checkpoint kinase 2 (p-Chk2) at Thr68, and total Chk2 (A) as well as xeroderma pigmentosum group D (XPD), xeroderma pigmentosum group A (XPA), DNA-binding protein 2 (DDB2), and DNA polymerase H (DNA Pol η) (B) in wild-type (WT) and p53-TAD knockout (KO) hiPSCs with and without doxorubicin (Doxo) treatment. Western blot quantifications are shown on the right. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. the respective nontreated cells; †P < 0.05 vs. WT; ‡P < 0.05 vs. WT + Doxo. C: p53-TAD hiPSCs were immunolabeled for γH2A.X (green; left) and counterstained with DAPI (blue; right). D: schematic of DNA damage repair methodology. E: nucleoids in WT hiPSCs and p53-TAD KO hiPSCs treated with Doxo (top) and after recovery (bottom) were stained with Vista green dye (green). Comets were apparent with Doxo and after recovery of p53-TAD KO hiPSCs, whereas intact DNA was noted in WT hiPSCs after recovery. F: tail moment of WT hiPSCs and p53-TAD KO hiPSCs nuclei at baseline (control), after Doxo, and after recovery. a.u., arbitrary units. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. control; **P < 0.05 vs. Doxo. G: representative micrograph of hiPSCs immunolabeled for γH2A.X (green; left) and nuclei stained with DAPI (blue; right). H: fraction of WT p53 and p53-TAD KO hiPSCs positive for γH2A.X. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. WT.

    Article Snippet: The following antibodies were used: NH 2 -terminal-specific rabbit monoclonal anti-p53 (Cell Signaling Technology, Danvers, MA), rabbit COOH-terminal anti-p53 (Sigma-Aldrich), rabbit monoclonal anti-checkpoint kinase 2 (Chk2; Thr 68 , Cell Signaling Technology), rabbit monoclonal anti-Chk2 (Cell Signaling Technology), rabbit monoclonal anti-Mre11 (Cell Signaling Technology), rabbit anti-Rad50 (Cell Signaling Technology), rabbit anti-xeroderma pigmentosum group D (XPD; D3Z6I, Cell Signaling Technology), rabbit anti-xeroderma pigmentosum group A (XPA; D9U5U, Cell Signaling Technology), rabbit anti-DNA polymerase H (DNA Pol η; E1I7T, Cell Signaling Technology), and rabbit anti-DNA-binding protein 2 (DDB2; D4C4, Cell Signaling Technology).

    Techniques: Western Blot, Binding Assay, Knock-Out, Immunolabeling, Staining