ddb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ddb2
    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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    ddb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ddb2
    Protection against UV-induced cytotoxicity by forced expression of <t>DDB2</t> . (A, B, C) Sensitivity to UV is presented for stable cell lines indicated or for (D, E, F) HR18 cells which express DDB2. The protein levels of DDB2, DDB1, and β-actin are shown in the insets. The plotted values represent means ± S.D. of experiments performed in triplicates. IC 50 values were also indicated in A and D.
    Ddb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Damaged DNA-binding protein 2 (DDB2) protects against UV irradiation in human cells and Drosophila"

    Article Title: Damaged DNA-binding protein 2 (DDB2) protects against UV irradiation in human cells and Drosophila

    Journal: Journal of Biomedical Science

    doi: 10.1186/1423-0127-17-27

    Protection against UV-induced cytotoxicity by forced expression of DDB2 . (A, B, C) Sensitivity to UV is presented for stable cell lines indicated or for (D, E, F) HR18 cells which express DDB2. The protein levels of DDB2, DDB1, and β-actin are shown in the insets. The plotted values represent means ± S.D. of experiments performed in triplicates. IC 50 values were also indicated in A and D.
    Figure Legend Snippet: Protection against UV-induced cytotoxicity by forced expression of DDB2 . (A, B, C) Sensitivity to UV is presented for stable cell lines indicated or for (D, E, F) HR18 cells which express DDB2. The protein levels of DDB2, DDB1, and β-actin are shown in the insets. The plotted values represent means ± S.D. of experiments performed in triplicates. IC 50 values were also indicated in A and D.

    Techniques Used: Expressing, Stable Transfection

    Overexpression of DDB2 is associated with reduced caspase activation following UV treatment . (A) Reduced caspase activation in cisplatin-resistant cells following UV irradiation. Cell extracts were immunoblotted with the antibodies indicated. (B) Reduced caspase activation in DDB2-expressing cells following UV irradiation.
    Figure Legend Snippet: Overexpression of DDB2 is associated with reduced caspase activation following UV treatment . (A) Reduced caspase activation in cisplatin-resistant cells following UV irradiation. Cell extracts were immunoblotted with the antibodies indicated. (B) Reduced caspase activation in DDB2-expressing cells following UV irradiation.

    Techniques Used: Over Expression, Activation Assay, Irradiation, Expressing

    Stimulation of cFLIP expression and attenuation of UV sensitivity by forced expression of DDB2 . (A) Overexpression of cFLIP in HR3 cells. The plotted values represent means ± S.D. of three experiments (right panel). (B) Stimulation of cFLIP expression by forced expression of DDB2. Whole cell extracts of HR18 cells, treated as indicated, were subjected to immunoblot analysis with specific antibodies. (C) Cell sensitivity to UV after Ad-DDB2 virus infection. The plotted values represent means ± S.D. of three experiments. ** Significant difference against control ( p < 0.05).
    Figure Legend Snippet: Stimulation of cFLIP expression and attenuation of UV sensitivity by forced expression of DDB2 . (A) Overexpression of cFLIP in HR3 cells. The plotted values represent means ± S.D. of three experiments (right panel). (B) Stimulation of cFLIP expression by forced expression of DDB2. Whole cell extracts of HR18 cells, treated as indicated, were subjected to immunoblot analysis with specific antibodies. (C) Cell sensitivity to UV after Ad-DDB2 virus infection. The plotted values represent means ± S.D. of three experiments. ** Significant difference against control ( p < 0.05).

    Techniques Used: Expressing, Over Expression, Western Blot, Infection

    Induction of endogenous cFLIP mRNA levels in cells following Ad-DDB2 infection.
    Figure Legend Snippet: Induction of endogenous cFLIP mRNA levels in cells following Ad-DDB2 infection.

    Techniques Used: Infection

    Upregulation of cFLIP promoter activity by forced expression of DDB2 . HEK293 cells were co-transfected with cFLIP reporter together with either control vector (pcDNA3) or DDB2-expressing vector (pcDNA3-DDB2). After 24 hrs, the luciferase activity was measured. The plotted values represent means ± S.D. from three independent transfections. The schematic presentation of full-length cFLIP promoter (-920/+43) and its deletion mutants are indicated below. Putative cis-elements are also indicated at positions relative to the transcription initiation site (+1). The construct number at the top indicates the length of the tested promoter region upstream of the putative transcription initiation site (designated by the bent arrow at +1). Luciferase activity is shown relative to the full-length cFLIP promoter (-920/+43). Significant difference to the control for each promoter is indicated.
    Figure Legend Snippet: Upregulation of cFLIP promoter activity by forced expression of DDB2 . HEK293 cells were co-transfected with cFLIP reporter together with either control vector (pcDNA3) or DDB2-expressing vector (pcDNA3-DDB2). After 24 hrs, the luciferase activity was measured. The plotted values represent means ± S.D. from three independent transfections. The schematic presentation of full-length cFLIP promoter (-920/+43) and its deletion mutants are indicated below. Putative cis-elements are also indicated at positions relative to the transcription initiation site (+1). The construct number at the top indicates the length of the tested promoter region upstream of the putative transcription initiation site (designated by the bent arrow at +1). Luciferase activity is shown relative to the full-length cFLIP promoter (-920/+43). Significant difference to the control for each promoter is indicated.

    Techniques Used: Activity Assay, Expressing, Transfection, Plasmid Preparation, Luciferase, Construct

    Resistance to UV in HR18 cells depends on DDB2 and cFLIP . (A) Attenuation of DDB2 protection against UV-induced apoptosis by knockdown of cFLIP using antisense oligonucleotides. HR18 cells were treated as described in the Materials and methods . The plotted values represent means ± S.D. of experiments performed in triplicates. Inset: immunoblot with specific antibodies. (B) Attenuation of UV-induced caspase activity by forced expression of DDB2, and resensitization by cFLIP antisense oligonucleotides.
    Figure Legend Snippet: Resistance to UV in HR18 cells depends on DDB2 and cFLIP . (A) Attenuation of DDB2 protection against UV-induced apoptosis by knockdown of cFLIP using antisense oligonucleotides. HR18 cells were treated as described in the Materials and methods . The plotted values represent means ± S.D. of experiments performed in triplicates. Inset: immunoblot with specific antibodies. (B) Attenuation of UV-induced caspase activity by forced expression of DDB2, and resensitization by cFLIP antisense oligonucleotides.

    Techniques Used: Western Blot, Activity Assay, Expressing

    Lack of attenuation of UV sensitivity by forced expression of DDB2 in cFLIP-lacking cells . (A) Lack of stimulation of cFLIP expression by forced expression of DDB2 in human VA13 and XP-A cells. Whole cell extracts of infected cells were compared. Cell extracts of cFLIP-transfected cells are also shown as cFLIP protein indicator. (B): Cell sensitivity to UV treatment after Ad-DDB2 virus infection in human VA13 and XP-A cells. The plotted values represent means ± S.D. of three experiments.
    Figure Legend Snippet: Lack of attenuation of UV sensitivity by forced expression of DDB2 in cFLIP-lacking cells . (A) Lack of stimulation of cFLIP expression by forced expression of DDB2 in human VA13 and XP-A cells. Whole cell extracts of infected cells were compared. Cell extracts of cFLIP-transfected cells are also shown as cFLIP protein indicator. (B): Cell sensitivity to UV treatment after Ad-DDB2 virus infection in human VA13 and XP-A cells. The plotted values represent means ± S.D. of three experiments.

    Techniques Used: Expressing, Infection, Transfection

    Protection against UV toxicity by DDB2 in Drosophila . (A) Expression of hDDB2-GFP in Drosophila . Protein extracts from GFP and GFP-hDDB2 expressing embryos were immunoblotted with anti-GFP antibodies. (B) Protection effect of hDDB2 against UV irradiation in fly larvae. GFP or hDDB2 overexpressing larvae were collected and then irradiated with UV (0-100 J/m 2 ). Four days later, surviving adults were counted and the survival rate was calculated. (n>4000). (C) Lack of inhibition of Reaper- or Eiger-induced apoptosis by hDDB2 over-expression. Eye morphology of wild-type and Reaper/eiger-overexpressing flies were examined. To analyze the effect of hDDB2, GMRGal4 was used to drive the expression of Reaper ( GMR>Rpr ) or Eiger ( GMR>eiger ) simultaneously with hDDB2 ( GMR>Rpr, UAS-hDDB2 and GMR>eiger, UAS-hDDB2 , respectively). Panel a-d: Reaper-induced apoptosis, resulting in small eyes, was not rescued by DDB2 overexpression. Panel a, Eye morphology of wild-type OreR fly; Panel b, Eye morphology of Rpr overexpressing fly; Panel c, Eye morphology of Rpr and DDB2 overexpressing fly; Panel d, Eye morphology of Rpr and P35 overexpressing fly. Panel e~g: Eiger-induced apoptosis not rescued by DDB2 overexpression. Panel e, Eye morphology of wild-type OreR fly; Panel f, Eye morphology of eiger overexpressing fly; Panel g, Eye morphology of eiger and DDB2 overexpressing fly.
    Figure Legend Snippet: Protection against UV toxicity by DDB2 in Drosophila . (A) Expression of hDDB2-GFP in Drosophila . Protein extracts from GFP and GFP-hDDB2 expressing embryos were immunoblotted with anti-GFP antibodies. (B) Protection effect of hDDB2 against UV irradiation in fly larvae. GFP or hDDB2 overexpressing larvae were collected and then irradiated with UV (0-100 J/m 2 ). Four days later, surviving adults were counted and the survival rate was calculated. (n>4000). (C) Lack of inhibition of Reaper- or Eiger-induced apoptosis by hDDB2 over-expression. Eye morphology of wild-type and Reaper/eiger-overexpressing flies were examined. To analyze the effect of hDDB2, GMRGal4 was used to drive the expression of Reaper ( GMR>Rpr ) or Eiger ( GMR>eiger ) simultaneously with hDDB2 ( GMR>Rpr, UAS-hDDB2 and GMR>eiger, UAS-hDDB2 , respectively). Panel a-d: Reaper-induced apoptosis, resulting in small eyes, was not rescued by DDB2 overexpression. Panel a, Eye morphology of wild-type OreR fly; Panel b, Eye morphology of Rpr overexpressing fly; Panel c, Eye morphology of Rpr and DDB2 overexpressing fly; Panel d, Eye morphology of Rpr and P35 overexpressing fly. Panel e~g: Eiger-induced apoptosis not rescued by DDB2 overexpression. Panel e, Eye morphology of wild-type OreR fly; Panel f, Eye morphology of eiger overexpressing fly; Panel g, Eye morphology of eiger and DDB2 overexpressing fly.

    Techniques Used: Expressing, Irradiation, Inhibition, Over Expression

    ddb2 proteo probe  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ddb2 proteo probe
    Ddb2 Proteo Probe, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, 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
    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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    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: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    ddb2  (Cell Signaling Technology Inc)


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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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    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.
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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
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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.
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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

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    anti ddb2  (Cell Signaling Technology Inc)
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    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.
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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