p53 acetyl lysine  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p53 acetyl lysine
    P53 Acetyl Lysine, 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
    https://www.bioz.com/result/p53 acetyl lysine/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
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    p53 acetyl lysine - by Bioz Stars, 2024-07
    86/100 stars

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    p53 acetyl lysine 382 specific antibodies  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc p53 acetyl lysine 382 specific antibodies
    P53 Acetyl Lysine 382 Specific Antibodies, 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
    https://www.bioz.com/result/p53 acetyl lysine 382 specific antibodies/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
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    86/100 stars

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    p53 lysine 164 acetylated antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p53 lysine 164 acetylated antibody
    ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on <t>p53</t> activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.
    P53 Lysine 164 Acetylated Antibody, 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
    https://www.bioz.com/result/p53 lysine 164 acetylated antibody/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p53 lysine 164 acetylated antibody - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "An unexpected role for Dicer as a reader of the unacetylated DNA binding domain of p53 in transcriptional regulation"

    Article Title: An unexpected role for Dicer as a reader of the unacetylated DNA binding domain of p53 in transcriptional regulation

    Journal: Science Advances

    doi: 10.1126/sciadv.abi6684

    ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.
    Figure Legend Snippet: ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.

    Techniques Used: Western Blot, CRISPR, Knock-Out, Clone Assay, Activity Assay, Negative Control, Positive Control, Transfection, Real-time Polymerase Chain Reaction

    ( A ) Schematic diagram of the p53 DBD. ( B ) Western blot analysis of Dicer and p53 DBD subdomains (DBD-N, amino acids 100 to 180; and DBD-C, amino acids 181 to 300) for their interaction in H1299 cells. ( C ) In vitro binding assay of biotin-conjugated K120 and K164 unacetylated or acetylated p53 peptides and Dicer. ( D ) Western blot analysis of Dicer and human p53 wild-type (WT), K120R, K164R, and 2KR (120/164) for their interaction in H1299 cells. ( E ) Western blot analysis of Dicer and mouse p53 WT and 3KR (117/161/162) for their interaction in H1299 cells. ( F ) Western blot analysis of the endogenous interaction between p53 and Dicer upon MG132 plus TSA/NAM treatment or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment of HCT116 cells. ( G ) Western blot analysis of p53 activity in H1299 mouse p53 WT, 3KR, or 3KR Dicer knockdown Tet-On cells with or without induction in time dependent manner. ( H and I ) Representative image and quantitative colony formation number in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 14 days. n = 3 for biological replicates. ( J ) Quantitative BrdU-positive ratio in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 24 hours. n = 8 biological replicable images. Error bars indicate means ± SD. All data are shown as representative of three experiments.
    Figure Legend Snippet: ( A ) Schematic diagram of the p53 DBD. ( B ) Western blot analysis of Dicer and p53 DBD subdomains (DBD-N, amino acids 100 to 180; and DBD-C, amino acids 181 to 300) for their interaction in H1299 cells. ( C ) In vitro binding assay of biotin-conjugated K120 and K164 unacetylated or acetylated p53 peptides and Dicer. ( D ) Western blot analysis of Dicer and human p53 wild-type (WT), K120R, K164R, and 2KR (120/164) for their interaction in H1299 cells. ( E ) Western blot analysis of Dicer and mouse p53 WT and 3KR (117/161/162) for their interaction in H1299 cells. ( F ) Western blot analysis of the endogenous interaction between p53 and Dicer upon MG132 plus TSA/NAM treatment or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment of HCT116 cells. ( G ) Western blot analysis of p53 activity in H1299 mouse p53 WT, 3KR, or 3KR Dicer knockdown Tet-On cells with or without induction in time dependent manner. ( H and I ) Representative image and quantitative colony formation number in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 14 days. n = 3 for biological replicates. ( J ) Quantitative BrdU-positive ratio in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 24 hours. n = 8 biological replicable images. Error bars indicate means ± SD. All data are shown as representative of three experiments.

    Techniques Used: Western Blot, In Vitro, Binding Assay, Activity Assay

    p53 lysine 120 acetylated antibody  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc p53 lysine 120 acetylated antibody
    ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on <t>p53</t> activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.
    P53 Lysine 120 Acetylated Antibody, 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
    https://www.bioz.com/result/p53 lysine 120 acetylated antibody/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p53 lysine 120 acetylated antibody - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "An unexpected role for Dicer as a reader of the unacetylated DNA binding domain of p53 in transcriptional regulation"

    Article Title: An unexpected role for Dicer as a reader of the unacetylated DNA binding domain of p53 in transcriptional regulation

    Journal: Science Advances

    doi: 10.1126/sciadv.abi6684

    ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.
    Figure Legend Snippet: ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.

    Techniques Used: Western Blot, CRISPR, Knock-Out, Clone Assay, Activity Assay, Negative Control, Positive Control, Transfection, Real-time Polymerase Chain Reaction

    ( A ) Schematic diagram of the p53 DBD. ( B ) Western blot analysis of Dicer and p53 DBD subdomains (DBD-N, amino acids 100 to 180; and DBD-C, amino acids 181 to 300) for their interaction in H1299 cells. ( C ) In vitro binding assay of biotin-conjugated K120 and K164 unacetylated or acetylated p53 peptides and Dicer. ( D ) Western blot analysis of Dicer and human p53 wild-type (WT), K120R, K164R, and 2KR (120/164) for their interaction in H1299 cells. ( E ) Western blot analysis of Dicer and mouse p53 WT and 3KR (117/161/162) for their interaction in H1299 cells. ( F ) Western blot analysis of the endogenous interaction between p53 and Dicer upon MG132 plus TSA/NAM treatment or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment of HCT116 cells. ( G ) Western blot analysis of p53 activity in H1299 mouse p53 WT, 3KR, or 3KR Dicer knockdown Tet-On cells with or without induction in time dependent manner. ( H and I ) Representative image and quantitative colony formation number in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 14 days. n = 3 for biological replicates. ( J ) Quantitative BrdU-positive ratio in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 24 hours. n = 8 biological replicable images. Error bars indicate means ± SD. All data are shown as representative of three experiments.
    Figure Legend Snippet: ( A ) Schematic diagram of the p53 DBD. ( B ) Western blot analysis of Dicer and p53 DBD subdomains (DBD-N, amino acids 100 to 180; and DBD-C, amino acids 181 to 300) for their interaction in H1299 cells. ( C ) In vitro binding assay of biotin-conjugated K120 and K164 unacetylated or acetylated p53 peptides and Dicer. ( D ) Western blot analysis of Dicer and human p53 wild-type (WT), K120R, K164R, and 2KR (120/164) for their interaction in H1299 cells. ( E ) Western blot analysis of Dicer and mouse p53 WT and 3KR (117/161/162) for their interaction in H1299 cells. ( F ) Western blot analysis of the endogenous interaction between p53 and Dicer upon MG132 plus TSA/NAM treatment or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment of HCT116 cells. ( G ) Western blot analysis of p53 activity in H1299 mouse p53 WT, 3KR, or 3KR Dicer knockdown Tet-On cells with or without induction in time dependent manner. ( H and I ) Representative image and quantitative colony formation number in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 14 days. n = 3 for biological replicates. ( J ) Quantitative BrdU-positive ratio in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 24 hours. n = 8 biological replicable images. Error bars indicate means ± SD. All data are shown as representative of three experiments.

    Techniques Used: Western Blot, In Vitro, Binding Assay, Activity Assay

    p53 lysine 382 acetylated antibody  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc p53 lysine 382 acetylated antibody
    ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on <t>p53</t> activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.
    P53 Lysine 382 Acetylated Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p53 lysine 382 acetylated antibody/product/Cell Signaling Technology Inc
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p53 lysine 382 acetylated antibody - by Bioz Stars, 2024-07
    96/100 stars

    Images

    1) Product Images from "An unexpected role for Dicer as a reader of the unacetylated DNA binding domain of p53 in transcriptional regulation"

    Article Title: An unexpected role for Dicer as a reader of the unacetylated DNA binding domain of p53 in transcriptional regulation

    Journal: Science Advances

    doi: 10.1126/sciadv.abi6684

    ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.
    Figure Legend Snippet: ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.

    Techniques Used: Western Blot, CRISPR, Knock-Out, Clone Assay, Activity Assay, Negative Control, Positive Control, Transfection, Real-time Polymerase Chain Reaction

    ( A ) Schematic diagram of the p53 DBD. ( B ) Western blot analysis of Dicer and p53 DBD subdomains (DBD-N, amino acids 100 to 180; and DBD-C, amino acids 181 to 300) for their interaction in H1299 cells. ( C ) In vitro binding assay of biotin-conjugated K120 and K164 unacetylated or acetylated p53 peptides and Dicer. ( D ) Western blot analysis of Dicer and human p53 wild-type (WT), K120R, K164R, and 2KR (120/164) for their interaction in H1299 cells. ( E ) Western blot analysis of Dicer and mouse p53 WT and 3KR (117/161/162) for their interaction in H1299 cells. ( F ) Western blot analysis of the endogenous interaction between p53 and Dicer upon MG132 plus TSA/NAM treatment or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment of HCT116 cells. ( G ) Western blot analysis of p53 activity in H1299 mouse p53 WT, 3KR, or 3KR Dicer knockdown Tet-On cells with or without induction in time dependent manner. ( H and I ) Representative image and quantitative colony formation number in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 14 days. n = 3 for biological replicates. ( J ) Quantitative BrdU-positive ratio in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 24 hours. n = 8 biological replicable images. Error bars indicate means ± SD. All data are shown as representative of three experiments.
    Figure Legend Snippet: ( A ) Schematic diagram of the p53 DBD. ( B ) Western blot analysis of Dicer and p53 DBD subdomains (DBD-N, amino acids 100 to 180; and DBD-C, amino acids 181 to 300) for their interaction in H1299 cells. ( C ) In vitro binding assay of biotin-conjugated K120 and K164 unacetylated or acetylated p53 peptides and Dicer. ( D ) Western blot analysis of Dicer and human p53 wild-type (WT), K120R, K164R, and 2KR (120/164) for their interaction in H1299 cells. ( E ) Western blot analysis of Dicer and mouse p53 WT and 3KR (117/161/162) for their interaction in H1299 cells. ( F ) Western blot analysis of the endogenous interaction between p53 and Dicer upon MG132 plus TSA/NAM treatment or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment of HCT116 cells. ( G ) Western blot analysis of p53 activity in H1299 mouse p53 WT, 3KR, or 3KR Dicer knockdown Tet-On cells with or without induction in time dependent manner. ( H and I ) Representative image and quantitative colony formation number in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 14 days. n = 3 for biological replicates. ( J ) Quantitative BrdU-positive ratio in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 24 hours. n = 8 biological replicable images. Error bars indicate means ± SD. All data are shown as representative of three experiments.

    Techniques Used: Western Blot, In Vitro, Binding Assay, Activity Assay

    p53 antibody  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc p53 antibody
    A. Silencing of SIRT1 by siRNA in AG11395 cell line by western blotting. B. In SIRT1 depleted WS cell, the expression level of acetylated <t>p53</t> (Ac-p53-K382) was checked by immunoblotting, and we found increased expression of Ac-p53-K382 in SIRT1 depleted WS cell. C. WS cells were treated with 10 μM resveratrol for 3 h followed by western blotting to analyze acetylated p53 and we observed reduction of Ac-p53-K382 in resveratrol treated WS cell. D. AG11395 cell line were transfected with WT WRN or acetylation mutants (K1127R, K1117R, K1127R/K1117R) prior to SIRT1 siRNA transfection after 24 h of incubation cells were treated with etoposide at 35 μM for 3 h. GFP foci were observed for 0 h and 24 h as recovery period but WT WRN able to relocate at nucleoli at 24 h in case of scramble siRNA transfection. At least 400–450 cells were counted for different condition under fluorescence microscope. E. Graphical representation of number of foci containing transfected WS cells during 0 h and 24 h of Etoposide treatment. More than 500 cells were counted for each condition. F. Cell with overexpressed acetylation mutants persists DSBs. Comet assay with AG11395 cells overexpressing either EGFPC1-WT WRN or mutants (K1127R, K1117R, K1127R/K1117R, empty vector) prior to SIRT1-siRNA plasmid transfection 24 h after 35 μM etoposide treatment. Represented images were taken by Komet Assay software. G. Graphical representation of etoposide treated transfected WS cells during 0 h and 24 h. The tail length was measured by Komet assay software. More than 18 cells were counted for each condition.
    P53 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p53 antibody/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p53 antibody - by Bioz Stars, 2024-07
    95/100 stars

    Images

    1) Product Images from "Acetylation of Werner protein at K1127 and K1117 is important for nuclear trafficking and DNA repair"

    Article Title: Acetylation of Werner protein at K1127 and K1117 is important for nuclear trafficking and DNA repair

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2019.04.010

    A. Silencing of SIRT1 by siRNA in AG11395 cell line by western blotting. B. In SIRT1 depleted WS cell, the expression level of acetylated p53 (Ac-p53-K382) was checked by immunoblotting, and we found increased expression of Ac-p53-K382 in SIRT1 depleted WS cell. C. WS cells were treated with 10 μM resveratrol for 3 h followed by western blotting to analyze acetylated p53 and we observed reduction of Ac-p53-K382 in resveratrol treated WS cell. D. AG11395 cell line were transfected with WT WRN or acetylation mutants (K1127R, K1117R, K1127R/K1117R) prior to SIRT1 siRNA transfection after 24 h of incubation cells were treated with etoposide at 35 μM for 3 h. GFP foci were observed for 0 h and 24 h as recovery period but WT WRN able to relocate at nucleoli at 24 h in case of scramble siRNA transfection. At least 400–450 cells were counted for different condition under fluorescence microscope. E. Graphical representation of number of foci containing transfected WS cells during 0 h and 24 h of Etoposide treatment. More than 500 cells were counted for each condition. F. Cell with overexpressed acetylation mutants persists DSBs. Comet assay with AG11395 cells overexpressing either EGFPC1-WT WRN or mutants (K1127R, K1117R, K1127R/K1117R, empty vector) prior to SIRT1-siRNA plasmid transfection 24 h after 35 μM etoposide treatment. Represented images were taken by Komet Assay software. G. Graphical representation of etoposide treated transfected WS cells during 0 h and 24 h. The tail length was measured by Komet assay software. More than 18 cells were counted for each condition.
    Figure Legend Snippet: A. Silencing of SIRT1 by siRNA in AG11395 cell line by western blotting. B. In SIRT1 depleted WS cell, the expression level of acetylated p53 (Ac-p53-K382) was checked by immunoblotting, and we found increased expression of Ac-p53-K382 in SIRT1 depleted WS cell. C. WS cells were treated with 10 μM resveratrol for 3 h followed by western blotting to analyze acetylated p53 and we observed reduction of Ac-p53-K382 in resveratrol treated WS cell. D. AG11395 cell line were transfected with WT WRN or acetylation mutants (K1127R, K1117R, K1127R/K1117R) prior to SIRT1 siRNA transfection after 24 h of incubation cells were treated with etoposide at 35 μM for 3 h. GFP foci were observed for 0 h and 24 h as recovery period but WT WRN able to relocate at nucleoli at 24 h in case of scramble siRNA transfection. At least 400–450 cells were counted for different condition under fluorescence microscope. E. Graphical representation of number of foci containing transfected WS cells during 0 h and 24 h of Etoposide treatment. More than 500 cells were counted for each condition. F. Cell with overexpressed acetylation mutants persists DSBs. Comet assay with AG11395 cells overexpressing either EGFPC1-WT WRN or mutants (K1127R, K1117R, K1127R/K1117R, empty vector) prior to SIRT1-siRNA plasmid transfection 24 h after 35 μM etoposide treatment. Represented images were taken by Komet Assay software. G. Graphical representation of etoposide treated transfected WS cells during 0 h and 24 h. The tail length was measured by Komet assay software. More than 18 cells were counted for each condition.

    Techniques Used: Western Blot, Expressing, Transfection, Incubation, Fluorescence, Microscopy, Single Cell Gel Electrophoresis, Plasmid Preparation, Software

    anti acetyl p53 lysine 382 antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti acetyl p53 lysine 382 antibodies
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    prid ab 331464 acetyl lysine 379 p53 cell signaling  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc acetyl lysine 379 p53
    Long-Term UV Exposure Depletes Epidermis of <t>p53</t> ∗/wt Cells (A) Protocol: Ahcre ERT -p53 ∗/wt mice were induced (green arrow), treated with a single dose of dimethylbenzanthracine (DMBA, black arrow), followed by repetitive sub-minimal erythema doses of UVB (red bars); blue arrows indicate sampling. (B) Confocal z stacks showing projected views of UV-irradiated and an adjacent unexposed area of IFE. Blue, DAPI; green, GFP, reporting p53 ∗ transcription; red, p53 indicating a p53 protein-stabilizing mutation. Scale bar, 20 μm. (C) Labeled projected area of p53 ∗/wt IFE in UV-exposed (purple circles; red line indicates mean value) and adjacent unexposed areas (green circles; red lines indicate mean value). n = 3 at the 12-week time point and n = 5 at the 28- to 36-week time point. Comparison between different time points, within same animal (unexposed versus UV irradiate area), ∗∗ p = 0.0046, ∗ p = 0.036 by unpaired two-tailed t test, ∗∗ p = 0.0071 by paired t test. (D) Proportion of labeled basal cells in UV-irradiated IFE at indicated time points. Values are mean percentage from 5–8 fields per animal. n = 3 mice per time point. Red bars, mean value. ∗ p = 0.042 by unpaired two-tailed t test. (E) Hypothesis: effect of prolonged low-dose UV on p53 ∗/wt clonal dynamics. Following induction, in the absence of UV, p53 ∗/wt clones (green) expand progressively in a background of wild-type cells (beige). In UV-exposed IFE, a wide variety of different mutant clones arises, indicated by multiple colors, some of which may expand, outcompete, and displace p53 ∗/wt cells from the IFE. (F) Simulation of clone competition under ongoing mutagenesis (see ). A transgenic mutant (green) is induced at 1% frequency in a background of wild-type cells (yellow). Subsequently, new mutations (red cross, shades of green if in transgenic cells, other colors if in wild-type cells) are induced at random and assigned a fitness value as described in . (G) The number of mutations detected per square millimeter by the Shearwater algorithm in each 16-mm 2 biopsy of mouse back skin: 3 induced and 2 non-induced mice at 12 weeks, 4 induced and 3 non-induced mice at 36 weeks. Solid circles, non-induced samples; open circles, induced samples. Green, non-exposed skin; purple, UV irradiated. ∗∗ p < 0.01, ∗ p = 0.02 by two-tailed Mann-Whitney test. Note that this method does not detect the induced and uninduced p53 ∗ allele. (H) Confocal z stacks showing p53 ∗/wt clone in direct contact with p53-immunopositive clone in UV-irradiated area (36 weeks post-induction). Blue, DAPI; green, GFP; red, p53. Scale bar, 20 μm. Dashed lines indicate outline of clones in top-down image and basement membrane in lateral view. Graph on the right shows proportion of p53 immunopositive area in UV-irradiated skin. Measurement of 8 fields of view per mouse are plotted individually. n = 3 mice per time point. Red lines indicate mean values. ∗∗∗∗ p < 0.0001 by two-tailed Mann-Whitney test. See also <xref ref-type=Figure S7 and and . " width="250" height="auto" />
    Acetyl Lysine 379 P53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Epidermal Tissue Adapts to Restrain Progenitors Carrying Clonal p53 Mutations"

    Article Title: Epidermal Tissue Adapts to Restrain Progenitors Carrying Clonal p53 Mutations

    Journal: Cell Stem Cell

    doi: 10.1016/j.stem.2018.08.017

    Long-Term UV Exposure Depletes Epidermis of p53 ∗/wt Cells (A) Protocol: Ahcre ERT -p53 ∗/wt mice were induced (green arrow), treated with a single dose of dimethylbenzanthracine (DMBA, black arrow), followed by repetitive sub-minimal erythema doses of UVB (red bars); blue arrows indicate sampling. (B) Confocal z stacks showing projected views of UV-irradiated and an adjacent unexposed area of IFE. Blue, DAPI; green, GFP, reporting p53 ∗ transcription; red, p53 indicating a p53 protein-stabilizing mutation. Scale bar, 20 μm. (C) Labeled projected area of p53 ∗/wt IFE in UV-exposed (purple circles; red line indicates mean value) and adjacent unexposed areas (green circles; red lines indicate mean value). n = 3 at the 12-week time point and n = 5 at the 28- to 36-week time point. Comparison between different time points, within same animal (unexposed versus UV irradiate area), ∗∗ p = 0.0046, ∗ p = 0.036 by unpaired two-tailed t test, ∗∗ p = 0.0071 by paired t test. (D) Proportion of labeled basal cells in UV-irradiated IFE at indicated time points. Values are mean percentage from 5–8 fields per animal. n = 3 mice per time point. Red bars, mean value. ∗ p = 0.042 by unpaired two-tailed t test. (E) Hypothesis: effect of prolonged low-dose UV on p53 ∗/wt clonal dynamics. Following induction, in the absence of UV, p53 ∗/wt clones (green) expand progressively in a background of wild-type cells (beige). In UV-exposed IFE, a wide variety of different mutant clones arises, indicated by multiple colors, some of which may expand, outcompete, and displace p53 ∗/wt cells from the IFE. (F) Simulation of clone competition under ongoing mutagenesis (see ). A transgenic mutant (green) is induced at 1% frequency in a background of wild-type cells (yellow). Subsequently, new mutations (red cross, shades of green if in transgenic cells, other colors if in wild-type cells) are induced at random and assigned a fitness value as described in . (G) The number of mutations detected per square millimeter by the Shearwater algorithm in each 16-mm 2 biopsy of mouse back skin: 3 induced and 2 non-induced mice at 12 weeks, 4 induced and 3 non-induced mice at 36 weeks. Solid circles, non-induced samples; open circles, induced samples. Green, non-exposed skin; purple, UV irradiated. ∗∗ p < 0.01, ∗ p = 0.02 by two-tailed Mann-Whitney test. Note that this method does not detect the induced and uninduced p53 ∗ allele. (H) Confocal z stacks showing p53 ∗/wt clone in direct contact with p53-immunopositive clone in UV-irradiated area (36 weeks post-induction). Blue, DAPI; green, GFP; red, p53. Scale bar, 20 μm. Dashed lines indicate outline of clones in top-down image and basement membrane in lateral view. Graph on the right shows proportion of p53 immunopositive area in UV-irradiated skin. Measurement of 8 fields of view per mouse are plotted individually. n = 3 mice per time point. Red lines indicate mean values. ∗∗∗∗ p < 0.0001 by two-tailed Mann-Whitney test. See also <xref ref-type=Figure S7 and and . " title="Long-Term UV Exposure Depletes Epidermis of p53 ∗/wt Cells (A) Protocol: Ahcre ERT -p53 ∗/wt ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Long-Term UV Exposure Depletes Epidermis of p53 ∗/wt Cells (A) Protocol: Ahcre ERT -p53 ∗/wt mice were induced (green arrow), treated with a single dose of dimethylbenzanthracine (DMBA, black arrow), followed by repetitive sub-minimal erythema doses of UVB (red bars); blue arrows indicate sampling. (B) Confocal z stacks showing projected views of UV-irradiated and an adjacent unexposed area of IFE. Blue, DAPI; green, GFP, reporting p53 ∗ transcription; red, p53 indicating a p53 protein-stabilizing mutation. Scale bar, 20 μm. (C) Labeled projected area of p53 ∗/wt IFE in UV-exposed (purple circles; red line indicates mean value) and adjacent unexposed areas (green circles; red lines indicate mean value). n = 3 at the 12-week time point and n = 5 at the 28- to 36-week time point. Comparison between different time points, within same animal (unexposed versus UV irradiate area), ∗∗ p = 0.0046, ∗ p = 0.036 by unpaired two-tailed t test, ∗∗ p = 0.0071 by paired t test. (D) Proportion of labeled basal cells in UV-irradiated IFE at indicated time points. Values are mean percentage from 5–8 fields per animal. n = 3 mice per time point. Red bars, mean value. ∗ p = 0.042 by unpaired two-tailed t test. (E) Hypothesis: effect of prolonged low-dose UV on p53 ∗/wt clonal dynamics. Following induction, in the absence of UV, p53 ∗/wt clones (green) expand progressively in a background of wild-type cells (beige). In UV-exposed IFE, a wide variety of different mutant clones arises, indicated by multiple colors, some of which may expand, outcompete, and displace p53 ∗/wt cells from the IFE. (F) Simulation of clone competition under ongoing mutagenesis (see ). A transgenic mutant (green) is induced at 1% frequency in a background of wild-type cells (yellow). Subsequently, new mutations (red cross, shades of green if in transgenic cells, other colors if in wild-type cells) are induced at random and assigned a fitness value as described in . (G) The number of mutations detected per square millimeter by the Shearwater algorithm in each 16-mm 2 biopsy of mouse back skin: 3 induced and 2 non-induced mice at 12 weeks, 4 induced and 3 non-induced mice at 36 weeks. Solid circles, non-induced samples; open circles, induced samples. Green, non-exposed skin; purple, UV irradiated. ∗∗ p < 0.01, ∗ p = 0.02 by two-tailed Mann-Whitney test. Note that this method does not detect the induced and uninduced p53 ∗ allele. (H) Confocal z stacks showing p53 ∗/wt clone in direct contact with p53-immunopositive clone in UV-irradiated area (36 weeks post-induction). Blue, DAPI; green, GFP; red, p53. Scale bar, 20 μm. Dashed lines indicate outline of clones in top-down image and basement membrane in lateral view. Graph on the right shows proportion of p53 immunopositive area in UV-irradiated skin. Measurement of 8 fields of view per mouse are plotted individually. n = 3 mice per time point. Red lines indicate mean values. ∗∗∗∗ p < 0.0001 by two-tailed Mann-Whitney test. See also Figure S7 and and .

    Techniques Used: Sampling, Irradiation, Mutagenesis, Labeling, Two Tailed Test, Clone Assay, Transgenic Assay, MANN-WHITNEY


    Figure Legend Snippet:

    Techniques Used: Plasmid Preparation, Recombinant, Acrylamide Gel Assay, Imaging, Western Blot, Staining, Purification, DNA Ligation, Immunoprecipitation, DNA Sequencing, Derivative Assay, TaqMan Assay, Software, Genome Wide, Variant Assay, Real-time Polymerase Chain Reaction

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    Cell Signaling Technology Inc acetyl lysine 379 p53
    Long-Term UV Exposure Depletes Epidermis of <t>p53</t> ∗/wt Cells (A) Protocol: Ahcre ERT -p53 ∗/wt mice were induced (green arrow), treated with a single dose of dimethylbenzanthracine (DMBA, black arrow), followed by repetitive sub-minimal erythema doses of UVB (red bars); blue arrows indicate sampling. (B) Confocal z stacks showing projected views of UV-irradiated and an adjacent unexposed area of IFE. Blue, DAPI; green, GFP, reporting p53 ∗ transcription; red, p53 indicating a p53 protein-stabilizing mutation. Scale bar, 20 μm. (C) Labeled projected area of p53 ∗/wt IFE in UV-exposed (purple circles; red line indicates mean value) and adjacent unexposed areas (green circles; red lines indicate mean value). n = 3 at the 12-week time point and n = 5 at the 28- to 36-week time point. Comparison between different time points, within same animal (unexposed versus UV irradiate area), ∗∗ p = 0.0046, ∗ p = 0.036 by unpaired two-tailed t test, ∗∗ p = 0.0071 by paired t test. (D) Proportion of labeled basal cells in UV-irradiated IFE at indicated time points. Values are mean percentage from 5–8 fields per animal. n = 3 mice per time point. Red bars, mean value. ∗ p = 0.042 by unpaired two-tailed t test. (E) Hypothesis: effect of prolonged low-dose UV on p53 ∗/wt clonal dynamics. Following induction, in the absence of UV, p53 ∗/wt clones (green) expand progressively in a background of wild-type cells (beige). In UV-exposed IFE, a wide variety of different mutant clones arises, indicated by multiple colors, some of which may expand, outcompete, and displace p53 ∗/wt cells from the IFE. (F) Simulation of clone competition under ongoing mutagenesis (see ). A transgenic mutant (green) is induced at 1% frequency in a background of wild-type cells (yellow). Subsequently, new mutations (red cross, shades of green if in transgenic cells, other colors if in wild-type cells) are induced at random and assigned a fitness value as described in . (G) The number of mutations detected per square millimeter by the Shearwater algorithm in each 16-mm 2 biopsy of mouse back skin: 3 induced and 2 non-induced mice at 12 weeks, 4 induced and 3 non-induced mice at 36 weeks. Solid circles, non-induced samples; open circles, induced samples. Green, non-exposed skin; purple, UV irradiated. ∗∗ p < 0.01, ∗ p = 0.02 by two-tailed Mann-Whitney test. Note that this method does not detect the induced and uninduced p53 ∗ allele. (H) Confocal z stacks showing p53 ∗/wt clone in direct contact with p53-immunopositive clone in UV-irradiated area (36 weeks post-induction). Blue, DAPI; green, GFP; red, p53. Scale bar, 20 μm. Dashed lines indicate outline of clones in top-down image and basement membrane in lateral view. Graph on the right shows proportion of p53 immunopositive area in UV-irradiated skin. Measurement of 8 fields of view per mouse are plotted individually. n = 3 mice per time point. Red lines indicate mean values. ∗∗∗∗ p < 0.0001 by two-tailed Mann-Whitney test. See also <xref ref-type=Figure S7 and and . " width="250" height="auto" />
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    ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.

    Journal: Science Advances

    Article Title: An unexpected role for Dicer as a reader of the unacetylated DNA binding domain of p53 in transcriptional regulation

    doi: 10.1126/sciadv.abi6684

    Figure Lengend Snippet: ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.

    Article Snippet: Information for antibodies is shown: p53 (DO-1; Santa Cruz Biotechnology, sc-126; IP, ChIP, or 1:1000 dilution for Western blot), p53 (CM5, Leica Biosystems; 1:1000 dilution), p53 (FL393; Bioss, bs-8687R; IP or ChIP), p-S15-p53 (Cell Signaling Technology, 9284; 1:1000 dilution), γH2AX (Cell Signaling Technology, 9718; 1:1000 dilution), Mdm2 (Ab-5; Millipore, OP-145; 1:500 dilution), p21 (12D1; Cell Signaling Technology, 2947; 1:1000 dilution), p21 (F-2; Santa Cruz Biotechnology, sc-6246; 1:250 dilution), PUMA (H-136; Santa Cruz Biotechnology, sc-28226; 1:500 dilution), Tigar (E-2; Santa Cruz Biotechnology, sc-166; 1:500 dilution), HA (3F10; Roche, 11867423001; ChIP or 1:2000 dilution for Western blot), myc (9E10; Santa Cruz Biotechnology, sc-40; 1:1000 dilution), Flag (Sigma-Aldrich, F3165; 1:5000 dilution), histone H3 acetyl K18 (Abcam, 1191; ChIP), histone H3 acetyl K27 (Abcam, 4729; ChIP), histone H3 trimethyl K27 (Abcam, 6002; ChIP), histone H3 monomethyl K4 (Abcam, 8895; ChIP), histone H3 trimethyl K9 (Abcam, 8898; ChIP), Vinculin (Sigma-Aldrich, V9131; 1:10,000 dilution), SUV39H1 (Upstate, 05-615; ChIP or 1:500 dilution for Western blot), Dicer (Bethyl, A301-936A; 1:1000 dilution), Dicer (Bethyl, A301-937A; IP), Dicer (Abcam, ab14601; 1:500 dilution), Drosha (Bethyl, A301-886A; 1:1000 dilution), DGCR8 (Bethyl, A302-468A; 1:1000 dilution), Lamin A/C (Bethyl, A303-430A; 1:10,000 dilution), actin (Sigma-Aldrich, A5441; 1:5000 dilution), 53BP1 (Upstate, 05-726; 1:500 dilution), USP28 (Bethyl, A300-898A; 1:2000 dilution), HP1α (Cell Signaling Technology, 2616; ChIP), and p53-lysine 382 acetylated antibody (Cell Signaling Technology, 2525; 1:500 dilution). p53-lysine 120 acetylated antibody (1:500 dilution), p53-lysine 164 acetylated antibody (1:500 dilution), or Sirt1 antibody (1:1000 dilution) was generated by our laboratory.

    Techniques: Western Blot, CRISPR, Knock-Out, Clone Assay, Activity Assay, Negative Control, Positive Control, Transfection, Real-time Polymerase Chain Reaction

    ( A ) Schematic diagram of the p53 DBD. ( B ) Western blot analysis of Dicer and p53 DBD subdomains (DBD-N, amino acids 100 to 180; and DBD-C, amino acids 181 to 300) for their interaction in H1299 cells. ( C ) In vitro binding assay of biotin-conjugated K120 and K164 unacetylated or acetylated p53 peptides and Dicer. ( D ) Western blot analysis of Dicer and human p53 wild-type (WT), K120R, K164R, and 2KR (120/164) for their interaction in H1299 cells. ( E ) Western blot analysis of Dicer and mouse p53 WT and 3KR (117/161/162) for their interaction in H1299 cells. ( F ) Western blot analysis of the endogenous interaction between p53 and Dicer upon MG132 plus TSA/NAM treatment or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment of HCT116 cells. ( G ) Western blot analysis of p53 activity in H1299 mouse p53 WT, 3KR, or 3KR Dicer knockdown Tet-On cells with or without induction in time dependent manner. ( H and I ) Representative image and quantitative colony formation number in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 14 days. n = 3 for biological replicates. ( J ) Quantitative BrdU-positive ratio in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 24 hours. n = 8 biological replicable images. Error bars indicate means ± SD. All data are shown as representative of three experiments.

    Journal: Science Advances

    Article Title: An unexpected role for Dicer as a reader of the unacetylated DNA binding domain of p53 in transcriptional regulation

    doi: 10.1126/sciadv.abi6684

    Figure Lengend Snippet: ( A ) Schematic diagram of the p53 DBD. ( B ) Western blot analysis of Dicer and p53 DBD subdomains (DBD-N, amino acids 100 to 180; and DBD-C, amino acids 181 to 300) for their interaction in H1299 cells. ( C ) In vitro binding assay of biotin-conjugated K120 and K164 unacetylated or acetylated p53 peptides and Dicer. ( D ) Western blot analysis of Dicer and human p53 wild-type (WT), K120R, K164R, and 2KR (120/164) for their interaction in H1299 cells. ( E ) Western blot analysis of Dicer and mouse p53 WT and 3KR (117/161/162) for their interaction in H1299 cells. ( F ) Western blot analysis of the endogenous interaction between p53 and Dicer upon MG132 plus TSA/NAM treatment or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment of HCT116 cells. ( G ) Western blot analysis of p53 activity in H1299 mouse p53 WT, 3KR, or 3KR Dicer knockdown Tet-On cells with or without induction in time dependent manner. ( H and I ) Representative image and quantitative colony formation number in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 14 days. n = 3 for biological replicates. ( J ) Quantitative BrdU-positive ratio in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 24 hours. n = 8 biological replicable images. Error bars indicate means ± SD. All data are shown as representative of three experiments.

    Article Snippet: Information for antibodies is shown: p53 (DO-1; Santa Cruz Biotechnology, sc-126; IP, ChIP, or 1:1000 dilution for Western blot), p53 (CM5, Leica Biosystems; 1:1000 dilution), p53 (FL393; Bioss, bs-8687R; IP or ChIP), p-S15-p53 (Cell Signaling Technology, 9284; 1:1000 dilution), γH2AX (Cell Signaling Technology, 9718; 1:1000 dilution), Mdm2 (Ab-5; Millipore, OP-145; 1:500 dilution), p21 (12D1; Cell Signaling Technology, 2947; 1:1000 dilution), p21 (F-2; Santa Cruz Biotechnology, sc-6246; 1:250 dilution), PUMA (H-136; Santa Cruz Biotechnology, sc-28226; 1:500 dilution), Tigar (E-2; Santa Cruz Biotechnology, sc-166; 1:500 dilution), HA (3F10; Roche, 11867423001; ChIP or 1:2000 dilution for Western blot), myc (9E10; Santa Cruz Biotechnology, sc-40; 1:1000 dilution), Flag (Sigma-Aldrich, F3165; 1:5000 dilution), histone H3 acetyl K18 (Abcam, 1191; ChIP), histone H3 acetyl K27 (Abcam, 4729; ChIP), histone H3 trimethyl K27 (Abcam, 6002; ChIP), histone H3 monomethyl K4 (Abcam, 8895; ChIP), histone H3 trimethyl K9 (Abcam, 8898; ChIP), Vinculin (Sigma-Aldrich, V9131; 1:10,000 dilution), SUV39H1 (Upstate, 05-615; ChIP or 1:500 dilution for Western blot), Dicer (Bethyl, A301-936A; 1:1000 dilution), Dicer (Bethyl, A301-937A; IP), Dicer (Abcam, ab14601; 1:500 dilution), Drosha (Bethyl, A301-886A; 1:1000 dilution), DGCR8 (Bethyl, A302-468A; 1:1000 dilution), Lamin A/C (Bethyl, A303-430A; 1:10,000 dilution), actin (Sigma-Aldrich, A5441; 1:5000 dilution), 53BP1 (Upstate, 05-726; 1:500 dilution), USP28 (Bethyl, A300-898A; 1:2000 dilution), HP1α (Cell Signaling Technology, 2616; ChIP), and p53-lysine 382 acetylated antibody (Cell Signaling Technology, 2525; 1:500 dilution). p53-lysine 120 acetylated antibody (1:500 dilution), p53-lysine 164 acetylated antibody (1:500 dilution), or Sirt1 antibody (1:1000 dilution) was generated by our laboratory.

    Techniques: Western Blot, In Vitro, Binding Assay, Activity Assay

    ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.

    Journal: Science Advances

    Article Title: An unexpected role for Dicer as a reader of the unacetylated DNA binding domain of p53 in transcriptional regulation

    doi: 10.1126/sciadv.abi6684

    Figure Lengend Snippet: ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.

    Article Snippet: Information for antibodies is shown: p53 (DO-1; Santa Cruz Biotechnology, sc-126; IP, ChIP, or 1:1000 dilution for Western blot), p53 (CM5, Leica Biosystems; 1:1000 dilution), p53 (FL393; Bioss, bs-8687R; IP or ChIP), p-S15-p53 (Cell Signaling Technology, 9284; 1:1000 dilution), γH2AX (Cell Signaling Technology, 9718; 1:1000 dilution), Mdm2 (Ab-5; Millipore, OP-145; 1:500 dilution), p21 (12D1; Cell Signaling Technology, 2947; 1:1000 dilution), p21 (F-2; Santa Cruz Biotechnology, sc-6246; 1:250 dilution), PUMA (H-136; Santa Cruz Biotechnology, sc-28226; 1:500 dilution), Tigar (E-2; Santa Cruz Biotechnology, sc-166; 1:500 dilution), HA (3F10; Roche, 11867423001; ChIP or 1:2000 dilution for Western blot), myc (9E10; Santa Cruz Biotechnology, sc-40; 1:1000 dilution), Flag (Sigma-Aldrich, F3165; 1:5000 dilution), histone H3 acetyl K18 (Abcam, 1191; ChIP), histone H3 acetyl K27 (Abcam, 4729; ChIP), histone H3 trimethyl K27 (Abcam, 6002; ChIP), histone H3 monomethyl K4 (Abcam, 8895; ChIP), histone H3 trimethyl K9 (Abcam, 8898; ChIP), Vinculin (Sigma-Aldrich, V9131; 1:10,000 dilution), SUV39H1 (Upstate, 05-615; ChIP or 1:500 dilution for Western blot), Dicer (Bethyl, A301-936A; 1:1000 dilution), Dicer (Bethyl, A301-937A; IP), Dicer (Abcam, ab14601; 1:500 dilution), Drosha (Bethyl, A301-886A; 1:1000 dilution), DGCR8 (Bethyl, A302-468A; 1:1000 dilution), Lamin A/C (Bethyl, A303-430A; 1:10,000 dilution), actin (Sigma-Aldrich, A5441; 1:5000 dilution), 53BP1 (Upstate, 05-726; 1:500 dilution), USP28 (Bethyl, A300-898A; 1:2000 dilution), HP1α (Cell Signaling Technology, 2616; ChIP), and p53-lysine 382 acetylated antibody (Cell Signaling Technology, 2525; 1:500 dilution). p53-lysine 120 acetylated antibody (1:500 dilution), p53-lysine 164 acetylated antibody (1:500 dilution), or Sirt1 antibody (1:1000 dilution) was generated by our laboratory.

    Techniques: Western Blot, CRISPR, Knock-Out, Clone Assay, Activity Assay, Negative Control, Positive Control, Transfection, Real-time Polymerase Chain Reaction

    ( A ) Schematic diagram of the p53 DBD. ( B ) Western blot analysis of Dicer and p53 DBD subdomains (DBD-N, amino acids 100 to 180; and DBD-C, amino acids 181 to 300) for their interaction in H1299 cells. ( C ) In vitro binding assay of biotin-conjugated K120 and K164 unacetylated or acetylated p53 peptides and Dicer. ( D ) Western blot analysis of Dicer and human p53 wild-type (WT), K120R, K164R, and 2KR (120/164) for their interaction in H1299 cells. ( E ) Western blot analysis of Dicer and mouse p53 WT and 3KR (117/161/162) for their interaction in H1299 cells. ( F ) Western blot analysis of the endogenous interaction between p53 and Dicer upon MG132 plus TSA/NAM treatment or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment of HCT116 cells. ( G ) Western blot analysis of p53 activity in H1299 mouse p53 WT, 3KR, or 3KR Dicer knockdown Tet-On cells with or without induction in time dependent manner. ( H and I ) Representative image and quantitative colony formation number in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 14 days. n = 3 for biological replicates. ( J ) Quantitative BrdU-positive ratio in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 24 hours. n = 8 biological replicable images. Error bars indicate means ± SD. All data are shown as representative of three experiments.

    Journal: Science Advances

    Article Title: An unexpected role for Dicer as a reader of the unacetylated DNA binding domain of p53 in transcriptional regulation

    doi: 10.1126/sciadv.abi6684

    Figure Lengend Snippet: ( A ) Schematic diagram of the p53 DBD. ( B ) Western blot analysis of Dicer and p53 DBD subdomains (DBD-N, amino acids 100 to 180; and DBD-C, amino acids 181 to 300) for their interaction in H1299 cells. ( C ) In vitro binding assay of biotin-conjugated K120 and K164 unacetylated or acetylated p53 peptides and Dicer. ( D ) Western blot analysis of Dicer and human p53 wild-type (WT), K120R, K164R, and 2KR (120/164) for their interaction in H1299 cells. ( E ) Western blot analysis of Dicer and mouse p53 WT and 3KR (117/161/162) for their interaction in H1299 cells. ( F ) Western blot analysis of the endogenous interaction between p53 and Dicer upon MG132 plus TSA/NAM treatment or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment of HCT116 cells. ( G ) Western blot analysis of p53 activity in H1299 mouse p53 WT, 3KR, or 3KR Dicer knockdown Tet-On cells with or without induction in time dependent manner. ( H and I ) Representative image and quantitative colony formation number in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 14 days. n = 3 for biological replicates. ( J ) Quantitative BrdU-positive ratio in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 24 hours. n = 8 biological replicable images. Error bars indicate means ± SD. All data are shown as representative of three experiments.

    Article Snippet: Information for antibodies is shown: p53 (DO-1; Santa Cruz Biotechnology, sc-126; IP, ChIP, or 1:1000 dilution for Western blot), p53 (CM5, Leica Biosystems; 1:1000 dilution), p53 (FL393; Bioss, bs-8687R; IP or ChIP), p-S15-p53 (Cell Signaling Technology, 9284; 1:1000 dilution), γH2AX (Cell Signaling Technology, 9718; 1:1000 dilution), Mdm2 (Ab-5; Millipore, OP-145; 1:500 dilution), p21 (12D1; Cell Signaling Technology, 2947; 1:1000 dilution), p21 (F-2; Santa Cruz Biotechnology, sc-6246; 1:250 dilution), PUMA (H-136; Santa Cruz Biotechnology, sc-28226; 1:500 dilution), Tigar (E-2; Santa Cruz Biotechnology, sc-166; 1:500 dilution), HA (3F10; Roche, 11867423001; ChIP or 1:2000 dilution for Western blot), myc (9E10; Santa Cruz Biotechnology, sc-40; 1:1000 dilution), Flag (Sigma-Aldrich, F3165; 1:5000 dilution), histone H3 acetyl K18 (Abcam, 1191; ChIP), histone H3 acetyl K27 (Abcam, 4729; ChIP), histone H3 trimethyl K27 (Abcam, 6002; ChIP), histone H3 monomethyl K4 (Abcam, 8895; ChIP), histone H3 trimethyl K9 (Abcam, 8898; ChIP), Vinculin (Sigma-Aldrich, V9131; 1:10,000 dilution), SUV39H1 (Upstate, 05-615; ChIP or 1:500 dilution for Western blot), Dicer (Bethyl, A301-936A; 1:1000 dilution), Dicer (Bethyl, A301-937A; IP), Dicer (Abcam, ab14601; 1:500 dilution), Drosha (Bethyl, A301-886A; 1:1000 dilution), DGCR8 (Bethyl, A302-468A; 1:1000 dilution), Lamin A/C (Bethyl, A303-430A; 1:10,000 dilution), actin (Sigma-Aldrich, A5441; 1:5000 dilution), 53BP1 (Upstate, 05-726; 1:500 dilution), USP28 (Bethyl, A300-898A; 1:2000 dilution), HP1α (Cell Signaling Technology, 2616; ChIP), and p53-lysine 382 acetylated antibody (Cell Signaling Technology, 2525; 1:500 dilution). p53-lysine 120 acetylated antibody (1:500 dilution), p53-lysine 164 acetylated antibody (1:500 dilution), or Sirt1 antibody (1:1000 dilution) was generated by our laboratory.

    Techniques: Western Blot, In Vitro, Binding Assay, Activity Assay

    ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.

    Journal: Science Advances

    Article Title: An unexpected role for Dicer as a reader of the unacetylated DNA binding domain of p53 in transcriptional regulation

    doi: 10.1126/sciadv.abi6684

    Figure Lengend Snippet: ( A ) Western blot analysis of the effect of different Dicer CRISPR-Cas9 knockout clones on p53 activity in HCT116 cells. sgRNA, single-guide RNA. ( B ) Western blot analysis of the effect of Dicer knockout in p53 +/+ or p53 −/− HCT116 cells. ( C ) Western blot analysis of the DNA damage response effect of Dicer knockout in p53 wild-type HCT116 cells. CRISPR-Cas9 negative control cells treated with doxorubicin (0.2 μg/ml) are positive control in DNA damage response. DMSO, dimethyl sulfoxide. sgNC, sgRNA Negative Control. ( D ) Western blot analysis of H1299 cells transfected with p53 or cotransfected with p53 and increasing amount of Dicer. ( E ) Quantitative polymerase chain reaction (qPCR) assay of the p21 , PUMA , and Mdm2 genes regulated by the p53 and/or Dicer transfection in H1299 cells. ( F and G ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer or Dicer-∆. ( H and I ) Western blot analysis and qPCR assay for detecting p21 level in H1299 cells transfected with p53 or cotransfected with p53 and Dicer CT or CT-D1320A. Error bars in qPCR assay indicate means ± SD. n = 3 technical replicates. All data are shown as representative of three experiments.

    Article Snippet: Information for antibodies is shown: p53 (DO-1; Santa Cruz Biotechnology, sc-126; IP, ChIP, or 1:1000 dilution for Western blot), p53 (CM5, Leica Biosystems; 1:1000 dilution), p53 (FL393; Bioss, bs-8687R; IP or ChIP), p-S15-p53 (Cell Signaling Technology, 9284; 1:1000 dilution), γH2AX (Cell Signaling Technology, 9718; 1:1000 dilution), Mdm2 (Ab-5; Millipore, OP-145; 1:500 dilution), p21 (12D1; Cell Signaling Technology, 2947; 1:1000 dilution), p21 (F-2; Santa Cruz Biotechnology, sc-6246; 1:250 dilution), PUMA (H-136; Santa Cruz Biotechnology, sc-28226; 1:500 dilution), Tigar (E-2; Santa Cruz Biotechnology, sc-166; 1:500 dilution), HA (3F10; Roche, 11867423001; ChIP or 1:2000 dilution for Western blot), myc (9E10; Santa Cruz Biotechnology, sc-40; 1:1000 dilution), Flag (Sigma-Aldrich, F3165; 1:5000 dilution), histone H3 acetyl K18 (Abcam, 1191; ChIP), histone H3 acetyl K27 (Abcam, 4729; ChIP), histone H3 trimethyl K27 (Abcam, 6002; ChIP), histone H3 monomethyl K4 (Abcam, 8895; ChIP), histone H3 trimethyl K9 (Abcam, 8898; ChIP), Vinculin (Sigma-Aldrich, V9131; 1:10,000 dilution), SUV39H1 (Upstate, 05-615; ChIP or 1:500 dilution for Western blot), Dicer (Bethyl, A301-936A; 1:1000 dilution), Dicer (Bethyl, A301-937A; IP), Dicer (Abcam, ab14601; 1:500 dilution), Drosha (Bethyl, A301-886A; 1:1000 dilution), DGCR8 (Bethyl, A302-468A; 1:1000 dilution), Lamin A/C (Bethyl, A303-430A; 1:10,000 dilution), actin (Sigma-Aldrich, A5441; 1:5000 dilution), 53BP1 (Upstate, 05-726; 1:500 dilution), USP28 (Bethyl, A300-898A; 1:2000 dilution), HP1α (Cell Signaling Technology, 2616; ChIP), and p53-lysine 382 acetylated antibody (Cell Signaling Technology, 2525; 1:500 dilution). p53-lysine 120 acetylated antibody (1:500 dilution), p53-lysine 164 acetylated antibody (1:500 dilution), or Sirt1 antibody (1:1000 dilution) was generated by our laboratory.

    Techniques: Western Blot, CRISPR, Knock-Out, Clone Assay, Activity Assay, Negative Control, Positive Control, Transfection, Real-time Polymerase Chain Reaction

    ( A ) Schematic diagram of the p53 DBD. ( B ) Western blot analysis of Dicer and p53 DBD subdomains (DBD-N, amino acids 100 to 180; and DBD-C, amino acids 181 to 300) for their interaction in H1299 cells. ( C ) In vitro binding assay of biotin-conjugated K120 and K164 unacetylated or acetylated p53 peptides and Dicer. ( D ) Western blot analysis of Dicer and human p53 wild-type (WT), K120R, K164R, and 2KR (120/164) for their interaction in H1299 cells. ( E ) Western blot analysis of Dicer and mouse p53 WT and 3KR (117/161/162) for their interaction in H1299 cells. ( F ) Western blot analysis of the endogenous interaction between p53 and Dicer upon MG132 plus TSA/NAM treatment or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment of HCT116 cells. ( G ) Western blot analysis of p53 activity in H1299 mouse p53 WT, 3KR, or 3KR Dicer knockdown Tet-On cells with or without induction in time dependent manner. ( H and I ) Representative image and quantitative colony formation number in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 14 days. n = 3 for biological replicates. ( J ) Quantitative BrdU-positive ratio in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 24 hours. n = 8 biological replicable images. Error bars indicate means ± SD. All data are shown as representative of three experiments.

    Journal: Science Advances

    Article Title: An unexpected role for Dicer as a reader of the unacetylated DNA binding domain of p53 in transcriptional regulation

    doi: 10.1126/sciadv.abi6684

    Figure Lengend Snippet: ( A ) Schematic diagram of the p53 DBD. ( B ) Western blot analysis of Dicer and p53 DBD subdomains (DBD-N, amino acids 100 to 180; and DBD-C, amino acids 181 to 300) for their interaction in H1299 cells. ( C ) In vitro binding assay of biotin-conjugated K120 and K164 unacetylated or acetylated p53 peptides and Dicer. ( D ) Western blot analysis of Dicer and human p53 wild-type (WT), K120R, K164R, and 2KR (120/164) for their interaction in H1299 cells. ( E ) Western blot analysis of Dicer and mouse p53 WT and 3KR (117/161/162) for their interaction in H1299 cells. ( F ) Western blot analysis of the endogenous interaction between p53 and Dicer upon MG132 plus TSA/NAM treatment or doxorubicin (0.2 μg/ml) plus TSA/NAM treatment of HCT116 cells. ( G ) Western blot analysis of p53 activity in H1299 mouse p53 WT, 3KR, or 3KR Dicer knockdown Tet-On cells with or without induction in time dependent manner. ( H and I ) Representative image and quantitative colony formation number in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 14 days. n = 3 for biological replicates. ( J ) Quantitative BrdU-positive ratio in H1299 mouse p53 3KR Tet-On sgNC or sgDicer cells with or without doxycycline (0.1 μg/ml) for 24 hours. n = 8 biological replicable images. Error bars indicate means ± SD. All data are shown as representative of three experiments.

    Article Snippet: Information for antibodies is shown: p53 (DO-1; Santa Cruz Biotechnology, sc-126; IP, ChIP, or 1:1000 dilution for Western blot), p53 (CM5, Leica Biosystems; 1:1000 dilution), p53 (FL393; Bioss, bs-8687R; IP or ChIP), p-S15-p53 (Cell Signaling Technology, 9284; 1:1000 dilution), γH2AX (Cell Signaling Technology, 9718; 1:1000 dilution), Mdm2 (Ab-5; Millipore, OP-145; 1:500 dilution), p21 (12D1; Cell Signaling Technology, 2947; 1:1000 dilution), p21 (F-2; Santa Cruz Biotechnology, sc-6246; 1:250 dilution), PUMA (H-136; Santa Cruz Biotechnology, sc-28226; 1:500 dilution), Tigar (E-2; Santa Cruz Biotechnology, sc-166; 1:500 dilution), HA (3F10; Roche, 11867423001; ChIP or 1:2000 dilution for Western blot), myc (9E10; Santa Cruz Biotechnology, sc-40; 1:1000 dilution), Flag (Sigma-Aldrich, F3165; 1:5000 dilution), histone H3 acetyl K18 (Abcam, 1191; ChIP), histone H3 acetyl K27 (Abcam, 4729; ChIP), histone H3 trimethyl K27 (Abcam, 6002; ChIP), histone H3 monomethyl K4 (Abcam, 8895; ChIP), histone H3 trimethyl K9 (Abcam, 8898; ChIP), Vinculin (Sigma-Aldrich, V9131; 1:10,000 dilution), SUV39H1 (Upstate, 05-615; ChIP or 1:500 dilution for Western blot), Dicer (Bethyl, A301-936A; 1:1000 dilution), Dicer (Bethyl, A301-937A; IP), Dicer (Abcam, ab14601; 1:500 dilution), Drosha (Bethyl, A301-886A; 1:1000 dilution), DGCR8 (Bethyl, A302-468A; 1:1000 dilution), Lamin A/C (Bethyl, A303-430A; 1:10,000 dilution), actin (Sigma-Aldrich, A5441; 1:5000 dilution), 53BP1 (Upstate, 05-726; 1:500 dilution), USP28 (Bethyl, A300-898A; 1:2000 dilution), HP1α (Cell Signaling Technology, 2616; ChIP), and p53-lysine 382 acetylated antibody (Cell Signaling Technology, 2525; 1:500 dilution). p53-lysine 120 acetylated antibody (1:500 dilution), p53-lysine 164 acetylated antibody (1:500 dilution), or Sirt1 antibody (1:1000 dilution) was generated by our laboratory.

    Techniques: Western Blot, In Vitro, Binding Assay, Activity Assay

    A. Silencing of SIRT1 by siRNA in AG11395 cell line by western blotting. B. In SIRT1 depleted WS cell, the expression level of acetylated p53 (Ac-p53-K382) was checked by immunoblotting, and we found increased expression of Ac-p53-K382 in SIRT1 depleted WS cell. C. WS cells were treated with 10 μM resveratrol for 3 h followed by western blotting to analyze acetylated p53 and we observed reduction of Ac-p53-K382 in resveratrol treated WS cell. D. AG11395 cell line were transfected with WT WRN or acetylation mutants (K1127R, K1117R, K1127R/K1117R) prior to SIRT1 siRNA transfection after 24 h of incubation cells were treated with etoposide at 35 μM for 3 h. GFP foci were observed for 0 h and 24 h as recovery period but WT WRN able to relocate at nucleoli at 24 h in case of scramble siRNA transfection. At least 400–450 cells were counted for different condition under fluorescence microscope. E. Graphical representation of number of foci containing transfected WS cells during 0 h and 24 h of Etoposide treatment. More than 500 cells were counted for each condition. F. Cell with overexpressed acetylation mutants persists DSBs. Comet assay with AG11395 cells overexpressing either EGFPC1-WT WRN or mutants (K1127R, K1117R, K1127R/K1117R, empty vector) prior to SIRT1-siRNA plasmid transfection 24 h after 35 μM etoposide treatment. Represented images were taken by Komet Assay software. G. Graphical representation of etoposide treated transfected WS cells during 0 h and 24 h. The tail length was measured by Komet assay software. More than 18 cells were counted for each condition.

    Journal: DNA repair

    Article Title: Acetylation of Werner protein at K1127 and K1117 is important for nuclear trafficking and DNA repair

    doi: 10.1016/j.dnarep.2019.04.010

    Figure Lengend Snippet: A. Silencing of SIRT1 by siRNA in AG11395 cell line by western blotting. B. In SIRT1 depleted WS cell, the expression level of acetylated p53 (Ac-p53-K382) was checked by immunoblotting, and we found increased expression of Ac-p53-K382 in SIRT1 depleted WS cell. C. WS cells were treated with 10 μM resveratrol for 3 h followed by western blotting to analyze acetylated p53 and we observed reduction of Ac-p53-K382 in resveratrol treated WS cell. D. AG11395 cell line were transfected with WT WRN or acetylation mutants (K1127R, K1117R, K1127R/K1117R) prior to SIRT1 siRNA transfection after 24 h of incubation cells were treated with etoposide at 35 μM for 3 h. GFP foci were observed for 0 h and 24 h as recovery period but WT WRN able to relocate at nucleoli at 24 h in case of scramble siRNA transfection. At least 400–450 cells were counted for different condition under fluorescence microscope. E. Graphical representation of number of foci containing transfected WS cells during 0 h and 24 h of Etoposide treatment. More than 500 cells were counted for each condition. F. Cell with overexpressed acetylation mutants persists DSBs. Comet assay with AG11395 cells overexpressing either EGFPC1-WT WRN or mutants (K1127R, K1117R, K1127R/K1117R, empty vector) prior to SIRT1-siRNA plasmid transfection 24 h after 35 μM etoposide treatment. Represented images were taken by Komet Assay software. G. Graphical representation of etoposide treated transfected WS cells during 0 h and 24 h. The tail length was measured by Komet assay software. More than 18 cells were counted for each condition.

    Article Snippet: Anti-β-actin, Anti-SIRT1 (CST # 2493S), Anti lysine (CST # 9681S), Anti-acetylated K382p53(# 2525 L) and anti p53 antibody were obtained from Cell Signaling Technologies.

    Techniques: Western Blot, Expressing, Transfection, Incubation, Fluorescence, Microscopy, Single Cell Gel Electrophoresis, Plasmid Preparation, Software

    Long-Term UV Exposure Depletes Epidermis of p53 ∗/wt Cells (A) Protocol: Ahcre ERT -p53 ∗/wt mice were induced (green arrow), treated with a single dose of dimethylbenzanthracine (DMBA, black arrow), followed by repetitive sub-minimal erythema doses of UVB (red bars); blue arrows indicate sampling. (B) Confocal z stacks showing projected views of UV-irradiated and an adjacent unexposed area of IFE. Blue, DAPI; green, GFP, reporting p53 ∗ transcription; red, p53 indicating a p53 protein-stabilizing mutation. Scale bar, 20 μm. (C) Labeled projected area of p53 ∗/wt IFE in UV-exposed (purple circles; red line indicates mean value) and adjacent unexposed areas (green circles; red lines indicate mean value). n = 3 at the 12-week time point and n = 5 at the 28- to 36-week time point. Comparison between different time points, within same animal (unexposed versus UV irradiate area), ∗∗ p = 0.0046, ∗ p = 0.036 by unpaired two-tailed t test, ∗∗ p = 0.0071 by paired t test. (D) Proportion of labeled basal cells in UV-irradiated IFE at indicated time points. Values are mean percentage from 5–8 fields per animal. n = 3 mice per time point. Red bars, mean value. ∗ p = 0.042 by unpaired two-tailed t test. (E) Hypothesis: effect of prolonged low-dose UV on p53 ∗/wt clonal dynamics. Following induction, in the absence of UV, p53 ∗/wt clones (green) expand progressively in a background of wild-type cells (beige). In UV-exposed IFE, a wide variety of different mutant clones arises, indicated by multiple colors, some of which may expand, outcompete, and displace p53 ∗/wt cells from the IFE. (F) Simulation of clone competition under ongoing mutagenesis (see ). A transgenic mutant (green) is induced at 1% frequency in a background of wild-type cells (yellow). Subsequently, new mutations (red cross, shades of green if in transgenic cells, other colors if in wild-type cells) are induced at random and assigned a fitness value as described in . (G) The number of mutations detected per square millimeter by the Shearwater algorithm in each 16-mm 2 biopsy of mouse back skin: 3 induced and 2 non-induced mice at 12 weeks, 4 induced and 3 non-induced mice at 36 weeks. Solid circles, non-induced samples; open circles, induced samples. Green, non-exposed skin; purple, UV irradiated. ∗∗ p < 0.01, ∗ p = 0.02 by two-tailed Mann-Whitney test. Note that this method does not detect the induced and uninduced p53 ∗ allele. (H) Confocal z stacks showing p53 ∗/wt clone in direct contact with p53-immunopositive clone in UV-irradiated area (36 weeks post-induction). Blue, DAPI; green, GFP; red, p53. Scale bar, 20 μm. Dashed lines indicate outline of clones in top-down image and basement membrane in lateral view. Graph on the right shows proportion of p53 immunopositive area in UV-irradiated skin. Measurement of 8 fields of view per mouse are plotted individually. n = 3 mice per time point. Red lines indicate mean values. ∗∗∗∗ p < 0.0001 by two-tailed Mann-Whitney test. See also <xref ref-type=Figure S7 and and . " width="100%" height="100%">

    Journal: Cell Stem Cell

    Article Title: Epidermal Tissue Adapts to Restrain Progenitors Carrying Clonal p53 Mutations

    doi: 10.1016/j.stem.2018.08.017

    Figure Lengend Snippet: Long-Term UV Exposure Depletes Epidermis of p53 ∗/wt Cells (A) Protocol: Ahcre ERT -p53 ∗/wt mice were induced (green arrow), treated with a single dose of dimethylbenzanthracine (DMBA, black arrow), followed by repetitive sub-minimal erythema doses of UVB (red bars); blue arrows indicate sampling. (B) Confocal z stacks showing projected views of UV-irradiated and an adjacent unexposed area of IFE. Blue, DAPI; green, GFP, reporting p53 ∗ transcription; red, p53 indicating a p53 protein-stabilizing mutation. Scale bar, 20 μm. (C) Labeled projected area of p53 ∗/wt IFE in UV-exposed (purple circles; red line indicates mean value) and adjacent unexposed areas (green circles; red lines indicate mean value). n = 3 at the 12-week time point and n = 5 at the 28- to 36-week time point. Comparison between different time points, within same animal (unexposed versus UV irradiate area), ∗∗ p = 0.0046, ∗ p = 0.036 by unpaired two-tailed t test, ∗∗ p = 0.0071 by paired t test. (D) Proportion of labeled basal cells in UV-irradiated IFE at indicated time points. Values are mean percentage from 5–8 fields per animal. n = 3 mice per time point. Red bars, mean value. ∗ p = 0.042 by unpaired two-tailed t test. (E) Hypothesis: effect of prolonged low-dose UV on p53 ∗/wt clonal dynamics. Following induction, in the absence of UV, p53 ∗/wt clones (green) expand progressively in a background of wild-type cells (beige). In UV-exposed IFE, a wide variety of different mutant clones arises, indicated by multiple colors, some of which may expand, outcompete, and displace p53 ∗/wt cells from the IFE. (F) Simulation of clone competition under ongoing mutagenesis (see ). A transgenic mutant (green) is induced at 1% frequency in a background of wild-type cells (yellow). Subsequently, new mutations (red cross, shades of green if in transgenic cells, other colors if in wild-type cells) are induced at random and assigned a fitness value as described in . (G) The number of mutations detected per square millimeter by the Shearwater algorithm in each 16-mm 2 biopsy of mouse back skin: 3 induced and 2 non-induced mice at 12 weeks, 4 induced and 3 non-induced mice at 36 weeks. Solid circles, non-induced samples; open circles, induced samples. Green, non-exposed skin; purple, UV irradiated. ∗∗ p < 0.01, ∗ p = 0.02 by two-tailed Mann-Whitney test. Note that this method does not detect the induced and uninduced p53 ∗ allele. (H) Confocal z stacks showing p53 ∗/wt clone in direct contact with p53-immunopositive clone in UV-irradiated area (36 weeks post-induction). Blue, DAPI; green, GFP; red, p53. Scale bar, 20 μm. Dashed lines indicate outline of clones in top-down image and basement membrane in lateral view. Graph on the right shows proportion of p53 immunopositive area in UV-irradiated skin. Measurement of 8 fields of view per mouse are plotted individually. n = 3 mice per time point. Red lines indicate mean values. ∗∗∗∗ p < 0.0001 by two-tailed Mann-Whitney test. See also Figure S7 and and .

    Article Snippet: Acetyl Lysine 379 p53 , Cell signaling , Cat# 2570; RRID: AB_823591.

    Techniques: Sampling, Irradiation, Mutagenesis, Labeling, Two Tailed Test, Clone Assay, Transgenic Assay, MANN-WHITNEY

    Journal: Cell Stem Cell

    Article Title: Epidermal Tissue Adapts to Restrain Progenitors Carrying Clonal p53 Mutations

    doi: 10.1016/j.stem.2018.08.017

    Figure Lengend Snippet:

    Article Snippet: Acetyl Lysine 379 p53 , Cell signaling , Cat# 2570; RRID: AB_823591.

    Techniques: Plasmid Preparation, Recombinant, Acrylamide Gel Assay, Imaging, Western Blot, Staining, Purification, DNA Ligation, Immunoprecipitation, DNA Sequencing, Derivative Assay, TaqMan Assay, Software, Genome Wide, Variant Assay, Real-time Polymerase Chain Reaction