53bp1 mcherry fragment Search Results


53bp1  (Addgene inc)
93
Addgene inc 53bp1
Histone signature in non-irradiated and irradiated cells without and with mutation or deletion in Suv39h1/h2 histone methyltransferases. Western blot analysis of γH2AX, H3K9me1, H3K9me2, H3K9me3, H3K9ac, H4K20me2, H4K20me3 and H4K20 acetylation. The levels of modified histones were normalized to that of total H3 histones. As DNA damage markers, <t>53BP1,</t> MDC1 proteins, and the HP1β protein were studied and normalized to the level of α-tubulin. Protein levels were studied in ( A ) HAP1 wt and HAP1 cells with the mutation in the SUV39H1 gene and ( B ) in wt MEFs and Suv39h1/h2-deficient fibroblasts (MEFs). Non-irradiated cells and cells irradiated by 5 Gy of γ-rays (harvested 30 min and 24 hours after irradiation) were analyzed. ( C ) Cell cycle profiles were studied by flow cytometry in non-irradiated and γ-irradiated wt MEFs and non-irradiated and γ-irradiated Suv39h1/h2 dn mouse embryonic fibroblasts. Panel ( a ) shows a 30-min interval, and panel ( b ) shows a 24-h interval when the cells were harvested after irradiation (and related control samples). Using Mod-Fit software, the percentage of cells in G1 (red peak), S (dash blue peak) and G2-M (green peak) cell cycle phases was calculated. The average cell cycle profile is shown for individual samples, and experiments were performed in 3 biological replicates.
53bp1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/53bp1+mcherry+fragment/pmc06224238-176-13-26?v=Addgene+inc
Average 93 stars, based on 1 article reviews
53bp1 - by Bioz Stars, 2026-07
93/100 stars
  Buy from Supplier
mcherry tagged 53bp1  (Addgene inc)
93
Addgene inc mcherry tagged 53bp1
Histone signature in non-irradiated and irradiated cells without and with mutation or deletion in Suv39h1/h2 histone methyltransferases. Western blot analysis of γH2AX, H3K9me1, H3K9me2, H3K9me3, H3K9ac, H4K20me2, H4K20me3 and H4K20 acetylation. The levels of modified histones were normalized to that of total H3 histones. As DNA damage markers, <t>53BP1,</t> MDC1 proteins, and the HP1β protein were studied and normalized to the level of α-tubulin. Protein levels were studied in ( A ) HAP1 wt and HAP1 cells with the mutation in the SUV39H1 gene and ( B ) in wt MEFs and Suv39h1/h2-deficient fibroblasts (MEFs). Non-irradiated cells and cells irradiated by 5 Gy of γ-rays (harvested 30 min and 24 hours after irradiation) were analyzed. ( C ) Cell cycle profiles were studied by flow cytometry in non-irradiated and γ-irradiated wt MEFs and non-irradiated and γ-irradiated Suv39h1/h2 dn mouse embryonic fibroblasts. Panel ( a ) shows a 30-min interval, and panel ( b ) shows a 24-h interval when the cells were harvested after irradiation (and related control samples). Using Mod-Fit software, the percentage of cells in G1 (red peak), S (dash blue peak) and G2-M (green peak) cell cycle phases was calculated. The average cell cycle profile is shown for individual samples, and experiments were performed in 3 biological replicates.
Mcherry Tagged 53bp1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/53bp1+mcherry+fragment/pmc06224238-176-12-26?v=Addgene+inc
Average 93 stars, based on 1 article reviews
mcherry tagged 53bp1 - by Bioz Stars, 2026-07
93/100 stars
  Buy from Supplier

Image Search Results


Histone signature in non-irradiated and irradiated cells without and with mutation or deletion in Suv39h1/h2 histone methyltransferases. Western blot analysis of γH2AX, H3K9me1, H3K9me2, H3K9me3, H3K9ac, H4K20me2, H4K20me3 and H4K20 acetylation. The levels of modified histones were normalized to that of total H3 histones. As DNA damage markers, 53BP1, MDC1 proteins, and the HP1β protein were studied and normalized to the level of α-tubulin. Protein levels were studied in ( A ) HAP1 wt and HAP1 cells with the mutation in the SUV39H1 gene and ( B ) in wt MEFs and Suv39h1/h2-deficient fibroblasts (MEFs). Non-irradiated cells and cells irradiated by 5 Gy of γ-rays (harvested 30 min and 24 hours after irradiation) were analyzed. ( C ) Cell cycle profiles were studied by flow cytometry in non-irradiated and γ-irradiated wt MEFs and non-irradiated and γ-irradiated Suv39h1/h2 dn mouse embryonic fibroblasts. Panel ( a ) shows a 30-min interval, and panel ( b ) shows a 24-h interval when the cells were harvested after irradiation (and related control samples). Using Mod-Fit software, the percentage of cells in G1 (red peak), S (dash blue peak) and G2-M (green peak) cell cycle phases was calculated. The average cell cycle profile is shown for individual samples, and experiments were performed in 3 biological replicates.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: Histone signature in non-irradiated and irradiated cells without and with mutation or deletion in Suv39h1/h2 histone methyltransferases. Western blot analysis of γH2AX, H3K9me1, H3K9me2, H3K9me3, H3K9ac, H4K20me2, H4K20me3 and H4K20 acetylation. The levels of modified histones were normalized to that of total H3 histones. As DNA damage markers, 53BP1, MDC1 proteins, and the HP1β protein were studied and normalized to the level of α-tubulin. Protein levels were studied in ( A ) HAP1 wt and HAP1 cells with the mutation in the SUV39H1 gene and ( B ) in wt MEFs and Suv39h1/h2-deficient fibroblasts (MEFs). Non-irradiated cells and cells irradiated by 5 Gy of γ-rays (harvested 30 min and 24 hours after irradiation) were analyzed. ( C ) Cell cycle profiles were studied by flow cytometry in non-irradiated and γ-irradiated wt MEFs and non-irradiated and γ-irradiated Suv39h1/h2 dn mouse embryonic fibroblasts. Panel ( a ) shows a 30-min interval, and panel ( b ) shows a 24-h interval when the cells were harvested after irradiation (and related control samples). Using Mod-Fit software, the percentage of cells in G1 (red peak), S (dash blue peak) and G2-M (green peak) cell cycle phases was calculated. The average cell cycle profile is shown for individual samples, and experiments were performed in 3 biological replicates.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Irradiation, Mutagenesis, Western Blot, Modification, Flow Cytometry, Software

Quantification of western blot data on selected histone markers in non-irradiated and γ-irradiated cells. Using ImageJ and ImageQuant TL software, the levels of the following proteins (originated from ) were quantified: H3K9me1, H3K9me2, H3K9me3, H3K9ac, H4K20me2, H4K20me3, H4K20ac, γH2AX, HP1β, 53BP1, and MDC1. The levels of modified histones were normalized to those of total H3 histones, and those of DDR-related proteins were normalized to those of α-tubulin. Quantification was performed in the following samples: ( A ) HAP1 wt and HAP1 mutant cells and ( B ) wt MEFs and Suv39h1/h2-deficient fibroblasts (MEFs).

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: Quantification of western blot data on selected histone markers in non-irradiated and γ-irradiated cells. Using ImageJ and ImageQuant TL software, the levels of the following proteins (originated from ) were quantified: H3K9me1, H3K9me2, H3K9me3, H3K9ac, H4K20me2, H4K20me3, H4K20ac, γH2AX, HP1β, 53BP1, and MDC1. The levels of modified histones were normalized to those of total H3 histones, and those of DDR-related proteins were normalized to those of α-tubulin. Quantification was performed in the following samples: ( A ) HAP1 wt and HAP1 mutant cells and ( B ) wt MEFs and Suv39h1/h2-deficient fibroblasts (MEFs).

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Western Blot, Irradiation, Software, Modification, Mutagenesis

The nuclear distribution pattern of 53BP1 protein. ( A ) Recruitment of 53BP1 to locally micro-irradiated chromatin in ( a ) Suv39h1/h2 wt and ( b ) Suv39h1/h2 dn fibroblasts. Quantification using LAS AF software in panel (b) shows that the level of the 53BP1 protein at UVA-irradiated ROIs was reduced in Suv39h1/h2 dn MEFs. For statistical analysis, the Student’s t-test was used; the difference in the protein levels is statistically significant at P≤0.05. ( B ) An average number of 53BP1-positive foci was significantly increased in γ-irradiated Suv39h1/h2 wt MEFs compared with those in the non-irradiated wt counterpart. In Suv39h1/h2 dn MEFs, γ-irradiation did not significantly change a number of 53BP1-positive repair foci. Student’s t-test was used for statistical analysis, and statistical significance was defined as P≤0.05. The distribution of 53BP1 in ( C ) in ( a ) non-irradiated and ( b ) γ-irradiated wild-type MEFs and ( D ) ( a ) non-irradiated and ( b ) γ-irradiated Suv39h1/h2 dn MEFs. Scale bars represent 10 µm.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: The nuclear distribution pattern of 53BP1 protein. ( A ) Recruitment of 53BP1 to locally micro-irradiated chromatin in ( a ) Suv39h1/h2 wt and ( b ) Suv39h1/h2 dn fibroblasts. Quantification using LAS AF software in panel (b) shows that the level of the 53BP1 protein at UVA-irradiated ROIs was reduced in Suv39h1/h2 dn MEFs. For statistical analysis, the Student’s t-test was used; the difference in the protein levels is statistically significant at P≤0.05. ( B ) An average number of 53BP1-positive foci was significantly increased in γ-irradiated Suv39h1/h2 wt MEFs compared with those in the non-irradiated wt counterpart. In Suv39h1/h2 dn MEFs, γ-irradiation did not significantly change a number of 53BP1-positive repair foci. Student’s t-test was used for statistical analysis, and statistical significance was defined as P≤0.05. The distribution of 53BP1 in ( C ) in ( a ) non-irradiated and ( b ) γ-irradiated wild-type MEFs and ( D ) ( a ) non-irradiated and ( b ) γ-irradiated Suv39h1/h2 dn MEFs. Scale bars represent 10 µm.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Irradiation, Software

The nuclear distribution pattern of H4K20me1/me2/me3 at DNA lesions. ( A ) The levels of ( a ) H4K20me1 (red) in γH2AX-positive DNA lesions (magenta), ( b ) H4K20me2 (red) in γH2AX-positive DNA lesions (magenta), and ( c ) H4K20me3 (red) in DNA lesions studied in parallel with γH2AX (magenta). ( B ) The level of ( a ) H4K20me3 and ( b ) 53BP1 in micro-irradiated ROI of the cells over-expressing JMJD2b histone demethylase, tagged by GFP. Panels Bb-aa show low level of 53BP1 at micro-irradiation induced DNA lesions in cells over-expressing GFP-tagged JMJD2b and panel Bb-bb documents 53BP1 recruitment to DSB sites in the cells with a normal expression of JMJD2b. Scale bars in all panels represent 5 µm.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: The nuclear distribution pattern of H4K20me1/me2/me3 at DNA lesions. ( A ) The levels of ( a ) H4K20me1 (red) in γH2AX-positive DNA lesions (magenta), ( b ) H4K20me2 (red) in γH2AX-positive DNA lesions (magenta), and ( c ) H4K20me3 (red) in DNA lesions studied in parallel with γH2AX (magenta). ( B ) The level of ( a ) H4K20me3 and ( b ) 53BP1 in micro-irradiated ROI of the cells over-expressing JMJD2b histone demethylase, tagged by GFP. Panels Bb-aa show low level of 53BP1 at micro-irradiation induced DNA lesions in cells over-expressing GFP-tagged JMJD2b and panel Bb-bb documents 53BP1 recruitment to DSB sites in the cells with a normal expression of JMJD2b. Scale bars in all panels represent 5 µm.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Irradiation, Expressing

The nuclear distribution pattern of H4K20me3 (green) in UV-damaged chromatin . ( Aa ) Non-irradiated and γ-irradiated Suv39h1/h2 wt and ( Ab ) non-irradiated and γ-irradiated Suv39h1/h2 dn MEFs. DAPI (blue) was used as a counterstain. Scale bars represent 10 µm. ( B ) Quantification of H4K20me3 (green) shown in panels Aa, Ab. Quantification of H4K20me3 was performed according to the selected region of interests (ROIs, yellow lines). LAS AX software was used for analysis of fluorescence intensities. ( C ) Nuclear distribution pattern of H4K20me3 in ( a ) non-irradiated and ( c ) γ-irradiated Suv39h1/h2 wt and ( b ) non-irradiated and ( d ) γ-irradiated Suv39h1/h2 dn fibroblasts. ( D ) Quantification of fluorescence intensity of H4K20me3 is shown in panel Ca-d. Analysis by LAS AF software was performed in ( a ) non-irradiated and ( c ) γ-irradiated Suv39h1/h2 wt and ( b ) non-irradiated and ( d ) γ-irradiated Suv39h1/h2 dn MEFs. ( E ) Nuclear distribution of H4K20me3 (green) and the 53BP1 protein (red) in ( a ) non-irradiated and ( c ) γ-irradiated Suv39h1/h2 wt and ( b ) non-irradiated and ( d ) γ-irradiated Suv39h1/h2 dn cells.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: The nuclear distribution pattern of H4K20me3 (green) in UV-damaged chromatin . ( Aa ) Non-irradiated and γ-irradiated Suv39h1/h2 wt and ( Ab ) non-irradiated and γ-irradiated Suv39h1/h2 dn MEFs. DAPI (blue) was used as a counterstain. Scale bars represent 10 µm. ( B ) Quantification of H4K20me3 (green) shown in panels Aa, Ab. Quantification of H4K20me3 was performed according to the selected region of interests (ROIs, yellow lines). LAS AX software was used for analysis of fluorescence intensities. ( C ) Nuclear distribution pattern of H4K20me3 in ( a ) non-irradiated and ( c ) γ-irradiated Suv39h1/h2 wt and ( b ) non-irradiated and ( d ) γ-irradiated Suv39h1/h2 dn fibroblasts. ( D ) Quantification of fluorescence intensity of H4K20me3 is shown in panel Ca-d. Analysis by LAS AF software was performed in ( a ) non-irradiated and ( c ) γ-irradiated Suv39h1/h2 wt and ( b ) non-irradiated and ( d ) γ-irradiated Suv39h1/h2 dn MEFs. ( E ) Nuclear distribution of H4K20me3 (green) and the 53BP1 protein (red) in ( a ) non-irradiated and ( c ) γ-irradiated Suv39h1/h2 wt and ( b ) non-irradiated and ( d ) γ-irradiated Suv39h1/h2 dn cells.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Irradiation, Software, Fluorescence

An interaction between 53BP1-H4K20me2/me3. ( A ) Immunoprecipitation (IP) experiments showed an interaction between H3K9me3 and 53BP1 or H4K20me2 and 53BP1 or H4K20me3 and the 53BP1 protein. HP1β protein did not interact with the 53BP1 protein. ( B ) Quantification of IP fragments from panel (A) studied in non-irradiated and γ-irradiated Suv39h1/h2 wt and Suv39h1/h2 dn cells. Asterisks (*) indicate statistical significance at P≤0.05.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: An interaction between 53BP1-H4K20me2/me3. ( A ) Immunoprecipitation (IP) experiments showed an interaction between H3K9me3 and 53BP1 or H4K20me2 and 53BP1 or H4K20me3 and the 53BP1 protein. HP1β protein did not interact with the 53BP1 protein. ( B ) Quantification of IP fragments from panel (A) studied in non-irradiated and γ-irradiated Suv39h1/h2 wt and Suv39h1/h2 dn cells. Asterisks (*) indicate statistical significance at P≤0.05.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Immunoprecipitation, Irradiation

Schematic illustration of a nucleosome with DSB occupied by studied proteins of interest in G1 and late S phase of the cell cycle. ( A ) Schema of an intact nucleosome. ( B ) Nucleosome with wound DNA with DSB. ( C ) A pictorial illustration of histone signature and recruitment of the 53BP1 protein to DNA lesions in the G1 phase of the cell cycle. Immunoprecipitation result shows dimerization of 53BP1 protein. ( D ) An illustration of histone signature and recruitment of PCNA and BRCA1 proteins to DNA lesions in late S phase of the cell cycle. Panels represent a schematic illustration of the results.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: Schematic illustration of a nucleosome with DSB occupied by studied proteins of interest in G1 and late S phase of the cell cycle. ( A ) Schema of an intact nucleosome. ( B ) Nucleosome with wound DNA with DSB. ( C ) A pictorial illustration of histone signature and recruitment of the 53BP1 protein to DNA lesions in the G1 phase of the cell cycle. Immunoprecipitation result shows dimerization of 53BP1 protein. ( D ) An illustration of histone signature and recruitment of PCNA and BRCA1 proteins to DNA lesions in late S phase of the cell cycle. Panels represent a schematic illustration of the results.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Immunoprecipitation

Schematic illustration of protein levels in DNA lesions in wild-type and Suv39h1/h2 dn mouse embryonic fibroblasts. Colored dots illustrate the levels of selected proteins at UVA-irradiated chromatin. The illustration demonstrates the appearance of the following proteins at DNA lesions: HP1β (pink circles), H3K9me3 (orange circles), H3K9ac (dark green circles), H4K20me3 (red circles), γH2AX (pale green circles), and 53BP1 (white circles). The selected micro-irradiated ROI is shown by a white rectangle. The figure represents an illustration of our results.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: Schematic illustration of protein levels in DNA lesions in wild-type and Suv39h1/h2 dn mouse embryonic fibroblasts. Colored dots illustrate the levels of selected proteins at UVA-irradiated chromatin. The illustration demonstrates the appearance of the following proteins at DNA lesions: HP1β (pink circles), H3K9me3 (orange circles), H3K9ac (dark green circles), H4K20me3 (red circles), γH2AX (pale green circles), and 53BP1 (white circles). The selected micro-irradiated ROI is shown by a white rectangle. The figure represents an illustration of our results.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Irradiation

Histone signature in non-irradiated and irradiated cells without and with mutation or deletion in Suv39h1/h2 histone methyltransferases. Western blot analysis of γH2AX, H3K9me1, H3K9me2, H3K9me3, H3K9ac, H4K20me2, H4K20me3 and H4K20 acetylation. The levels of modified histones were normalized to that of total H3 histones. As DNA damage markers, 53BP1, MDC1 proteins, and the HP1β protein were studied and normalized to the level of α-tubulin. Protein levels were studied in ( A ) HAP1 wt and HAP1 cells with the mutation in the SUV39H1 gene and ( B ) in wt MEFs and Suv39h1/h2-deficient fibroblasts (MEFs). Non-irradiated cells and cells irradiated by 5 Gy of γ-rays (harvested 30 min and 24 hours after irradiation) were analyzed. ( C ) Cell cycle profiles were studied by flow cytometry in non-irradiated and γ-irradiated wt MEFs and non-irradiated and γ-irradiated Suv39h1/h2 dn mouse embryonic fibroblasts. Panel ( a ) shows a 30-min interval, and panel ( b ) shows a 24-h interval when the cells were harvested after irradiation (and related control samples). Using Mod-Fit software, the percentage of cells in G1 (red peak), S (dash blue peak) and G2-M (green peak) cell cycle phases was calculated. The average cell cycle profile is shown for individual samples, and experiments were performed in 3 biological replicates.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: Histone signature in non-irradiated and irradiated cells without and with mutation or deletion in Suv39h1/h2 histone methyltransferases. Western blot analysis of γH2AX, H3K9me1, H3K9me2, H3K9me3, H3K9ac, H4K20me2, H4K20me3 and H4K20 acetylation. The levels of modified histones were normalized to that of total H3 histones. As DNA damage markers, 53BP1, MDC1 proteins, and the HP1β protein were studied and normalized to the level of α-tubulin. Protein levels were studied in ( A ) HAP1 wt and HAP1 cells with the mutation in the SUV39H1 gene and ( B ) in wt MEFs and Suv39h1/h2-deficient fibroblasts (MEFs). Non-irradiated cells and cells irradiated by 5 Gy of γ-rays (harvested 30 min and 24 hours after irradiation) were analyzed. ( C ) Cell cycle profiles were studied by flow cytometry in non-irradiated and γ-irradiated wt MEFs and non-irradiated and γ-irradiated Suv39h1/h2 dn mouse embryonic fibroblasts. Panel ( a ) shows a 30-min interval, and panel ( b ) shows a 24-h interval when the cells were harvested after irradiation (and related control samples). Using Mod-Fit software, the percentage of cells in G1 (red peak), S (dash blue peak) and G2-M (green peak) cell cycle phases was calculated. The average cell cycle profile is shown for individual samples, and experiments were performed in 3 biological replicates.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Irradiation, Mutagenesis, Western Blot, Modification, Flow Cytometry, Control, Software

Quantification of western blot data on selected histone markers in non-irradiated and γ-irradiated cells. Using ImageJ and ImageQuant TL software, the levels of the following proteins (originated from ) were quantified: H3K9me1, H3K9me2, H3K9me3, H3K9ac, H4K20me2, H4K20me3, H4K20ac, γH2AX, HP1β, 53BP1, and MDC1. The levels of modified histones were normalized to those of total H3 histones, and those of DDR-related proteins were normalized to those of α-tubulin. Quantification was performed in the following samples: ( A ) HAP1 wt and HAP1 mutant cells and ( B ) wt MEFs and Suv39h1/h2-deficient fibroblasts (MEFs).

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: Quantification of western blot data on selected histone markers in non-irradiated and γ-irradiated cells. Using ImageJ and ImageQuant TL software, the levels of the following proteins (originated from ) were quantified: H3K9me1, H3K9me2, H3K9me3, H3K9ac, H4K20me2, H4K20me3, H4K20ac, γH2AX, HP1β, 53BP1, and MDC1. The levels of modified histones were normalized to those of total H3 histones, and those of DDR-related proteins were normalized to those of α-tubulin. Quantification was performed in the following samples: ( A ) HAP1 wt and HAP1 mutant cells and ( B ) wt MEFs and Suv39h1/h2-deficient fibroblasts (MEFs).

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Western Blot, Irradiation, Software, Modification, Mutagenesis

The nuclear distribution pattern of 53BP1 protein. ( A ) Recruitment of 53BP1 to locally micro-irradiated chromatin in ( a ) Suv39h1/h2 wt and ( b ) Suv39h1/h2 dn fibroblasts. Quantification using LAS AF software in panel (b) shows that the level of the 53BP1 protein at UVA-irradiated ROIs was reduced in Suv39h1/h2 dn MEFs. For statistical analysis, the Student’s t-test was used; the difference in the protein levels is statistically significant at P≤0.05. ( B ) An average number of 53BP1-positive foci was significantly increased in γ-irradiated Suv39h1/h2 wt MEFs compared with those in the non-irradiated wt counterpart. In Suv39h1/h2 dn MEFs, γ-irradiation did not significantly change a number of 53BP1-positive repair foci. Student’s t-test was used for statistical analysis, and statistical significance was defined as P≤0.05. The distribution of 53BP1 in ( C ) in ( a ) non-irradiated and ( b ) γ-irradiated wild-type MEFs and ( D ) ( a ) non-irradiated and ( b ) γ-irradiated Suv39h1/h2 dn MEFs. Scale bars represent 10 µm.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: The nuclear distribution pattern of 53BP1 protein. ( A ) Recruitment of 53BP1 to locally micro-irradiated chromatin in ( a ) Suv39h1/h2 wt and ( b ) Suv39h1/h2 dn fibroblasts. Quantification using LAS AF software in panel (b) shows that the level of the 53BP1 protein at UVA-irradiated ROIs was reduced in Suv39h1/h2 dn MEFs. For statistical analysis, the Student’s t-test was used; the difference in the protein levels is statistically significant at P≤0.05. ( B ) An average number of 53BP1-positive foci was significantly increased in γ-irradiated Suv39h1/h2 wt MEFs compared with those in the non-irradiated wt counterpart. In Suv39h1/h2 dn MEFs, γ-irradiation did not significantly change a number of 53BP1-positive repair foci. Student’s t-test was used for statistical analysis, and statistical significance was defined as P≤0.05. The distribution of 53BP1 in ( C ) in ( a ) non-irradiated and ( b ) γ-irradiated wild-type MEFs and ( D ) ( a ) non-irradiated and ( b ) γ-irradiated Suv39h1/h2 dn MEFs. Scale bars represent 10 µm.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Irradiation, Software

The nuclear distribution pattern of H4K20me1/me2/me3 at DNA lesions. ( A ) The levels of ( a ) H4K20me1 (red) in γH2AX-positive DNA lesions (magenta), ( b ) H4K20me2 (red) in γH2AX-positive DNA lesions (magenta), and ( c ) H4K20me3 (red) in DNA lesions studied in parallel with γH2AX (magenta). ( B ) The level of ( a ) H4K20me3 and ( b ) 53BP1 in micro-irradiated ROI of the cells over-expressing JMJD2b histone demethylase, tagged by GFP. Panels Bb-aa show low level of 53BP1 at micro-irradiation induced DNA lesions in cells over-expressing GFP-tagged JMJD2b and panel Bb-bb documents 53BP1 recruitment to DSB sites in the cells with a normal expression of JMJD2b. Scale bars in all panels represent 5 µm.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: The nuclear distribution pattern of H4K20me1/me2/me3 at DNA lesions. ( A ) The levels of ( a ) H4K20me1 (red) in γH2AX-positive DNA lesions (magenta), ( b ) H4K20me2 (red) in γH2AX-positive DNA lesions (magenta), and ( c ) H4K20me3 (red) in DNA lesions studied in parallel with γH2AX (magenta). ( B ) The level of ( a ) H4K20me3 and ( b ) 53BP1 in micro-irradiated ROI of the cells over-expressing JMJD2b histone demethylase, tagged by GFP. Panels Bb-aa show low level of 53BP1 at micro-irradiation induced DNA lesions in cells over-expressing GFP-tagged JMJD2b and panel Bb-bb documents 53BP1 recruitment to DSB sites in the cells with a normal expression of JMJD2b. Scale bars in all panels represent 5 µm.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Irradiation, Expressing

The nuclear distribution pattern of H4K20me3 (green) in UV-damaged chromatin . ( Aa ) Non-irradiated and γ-irradiated Suv39h1/h2 wt and ( Ab ) non-irradiated and γ-irradiated Suv39h1/h2 dn MEFs. DAPI (blue) was used as a counterstain. Scale bars represent 10 µm. ( B ) Quantification of H4K20me3 (green) shown in panels Aa, Ab. Quantification of H4K20me3 was performed according to the selected region of interests (ROIs, yellow lines). LAS AX software was used for analysis of fluorescence intensities. ( C ) Nuclear distribution pattern of H4K20me3 in ( a ) non-irradiated and ( c ) γ-irradiated Suv39h1/h2 wt and ( b ) non-irradiated and ( d ) γ-irradiated Suv39h1/h2 dn fibroblasts. ( D ) Quantification of fluorescence intensity of H4K20me3 is shown in panel Ca-d. Analysis by LAS AF software was performed in ( a ) non-irradiated and ( c ) γ-irradiated Suv39h1/h2 wt and ( b ) non-irradiated and ( d ) γ-irradiated Suv39h1/h2 dn MEFs. ( E ) Nuclear distribution of H4K20me3 (green) and the 53BP1 protein (red) in ( a ) non-irradiated and ( c ) γ-irradiated Suv39h1/h2 wt and ( b ) non-irradiated and ( d ) γ-irradiated Suv39h1/h2 dn cells.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: The nuclear distribution pattern of H4K20me3 (green) in UV-damaged chromatin . ( Aa ) Non-irradiated and γ-irradiated Suv39h1/h2 wt and ( Ab ) non-irradiated and γ-irradiated Suv39h1/h2 dn MEFs. DAPI (blue) was used as a counterstain. Scale bars represent 10 µm. ( B ) Quantification of H4K20me3 (green) shown in panels Aa, Ab. Quantification of H4K20me3 was performed according to the selected region of interests (ROIs, yellow lines). LAS AX software was used for analysis of fluorescence intensities. ( C ) Nuclear distribution pattern of H4K20me3 in ( a ) non-irradiated and ( c ) γ-irradiated Suv39h1/h2 wt and ( b ) non-irradiated and ( d ) γ-irradiated Suv39h1/h2 dn fibroblasts. ( D ) Quantification of fluorescence intensity of H4K20me3 is shown in panel Ca-d. Analysis by LAS AF software was performed in ( a ) non-irradiated and ( c ) γ-irradiated Suv39h1/h2 wt and ( b ) non-irradiated and ( d ) γ-irradiated Suv39h1/h2 dn MEFs. ( E ) Nuclear distribution of H4K20me3 (green) and the 53BP1 protein (red) in ( a ) non-irradiated and ( c ) γ-irradiated Suv39h1/h2 wt and ( b ) non-irradiated and ( d ) γ-irradiated Suv39h1/h2 dn cells.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Irradiation, Software, Fluorescence

An interaction between 53BP1-H4K20me2/me3. ( A ) Immunoprecipitation (IP) experiments showed an interaction between H3K9me3 and 53BP1 or H4K20me2 and 53BP1 or H4K20me3 and the 53BP1 protein. HP1β protein did not interact with the 53BP1 protein. ( B ) Quantification of IP fragments from panel (A) studied in non-irradiated and γ-irradiated Suv39h1/h2 wt and Suv39h1/h2 dn cells. Asterisks (*) indicate statistical significance at P≤0.05.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: An interaction between 53BP1-H4K20me2/me3. ( A ) Immunoprecipitation (IP) experiments showed an interaction between H3K9me3 and 53BP1 or H4K20me2 and 53BP1 or H4K20me3 and the 53BP1 protein. HP1β protein did not interact with the 53BP1 protein. ( B ) Quantification of IP fragments from panel (A) studied in non-irradiated and γ-irradiated Suv39h1/h2 wt and Suv39h1/h2 dn cells. Asterisks (*) indicate statistical significance at P≤0.05.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Immunoprecipitation, Irradiation

Schematic illustration of a nucleosome with DSB occupied by studied proteins of interest in G1 and late S phase of the cell cycle. ( A ) Schema of an intact nucleosome. ( B ) Nucleosome with wound DNA with DSB. ( C ) A pictorial illustration of histone signature and recruitment of the 53BP1 protein to DNA lesions in the G1 phase of the cell cycle. Immunoprecipitation result shows dimerization of 53BP1 protein. ( D ) An illustration of histone signature and recruitment of PCNA and BRCA1 proteins to DNA lesions in late S phase of the cell cycle. Panels represent a schematic illustration of the results.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: Schematic illustration of a nucleosome with DSB occupied by studied proteins of interest in G1 and late S phase of the cell cycle. ( A ) Schema of an intact nucleosome. ( B ) Nucleosome with wound DNA with DSB. ( C ) A pictorial illustration of histone signature and recruitment of the 53BP1 protein to DNA lesions in the G1 phase of the cell cycle. Immunoprecipitation result shows dimerization of 53BP1 protein. ( D ) An illustration of histone signature and recruitment of PCNA and BRCA1 proteins to DNA lesions in late S phase of the cell cycle. Panels represent a schematic illustration of the results.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Immunoprecipitation

Schematic illustration of protein levels in DNA lesions in wild-type and Suv39h1/h2 dn mouse embryonic fibroblasts. Colored dots illustrate the levels of selected proteins at UVA-irradiated chromatin. The illustration demonstrates the appearance of the following proteins at DNA lesions: HP1β (pink circles), H3K9me3 (orange circles), H3K9ac (dark green circles), H4K20me3 (red circles), γH2AX (pale green circles), and 53BP1 (white circles). The selected micro-irradiated ROI is shown by a white rectangle. The figure represents an illustration of our results.

Journal: Aging (Albany NY)

Article Title: H3K9me3 and H4K20me3 represent the epigenetic landscape for 53BP1 binding to DNA lesions

doi: 10.18632/aging.101572

Figure Lengend Snippet: Schematic illustration of protein levels in DNA lesions in wild-type and Suv39h1/h2 dn mouse embryonic fibroblasts. Colored dots illustrate the levels of selected proteins at UVA-irradiated chromatin. The illustration demonstrates the appearance of the following proteins at DNA lesions: HP1β (pink circles), H3K9me3 (orange circles), H3K9ac (dark green circles), H4K20me3 (red circles), γH2AX (pale green circles), and 53BP1 (white circles). The selected micro-irradiated ROI is shown by a white rectangle. The figure represents an illustration of our results.

Article Snippet: For live-cell studies, we used the following plasmids: GFP-tagged HP1β [ ]; mCherry-tagged 53BP1 (mCherry-BP1-2 pLPC-Puro (a fragment of human 53BP1, aa 1220 - 1711; #19835, Addgene, Cambridge, Massachusetts, USA), mCherry-tagged PCNA (a generous gift from prof. Christina Cardoso, Technical University, Darmstadt, Germany), pDEST-FRT/T0-GFP-BRCA1 (#71116, Addgene, USA), and GFP-tagged JMJD2b (termed GFP-JMJD2b-1086), (a generous gift from prof. Thomas Jenuwein and Dr. Nicholas Shukeir, Max Planck Institute of Immunobiology, Freiburg, Germany).

Techniques: Irradiation