mouse anti γh2ax  (Novus Biologicals)


Bioz Verified Symbol Novus Biologicals is a verified supplier
Bioz Manufacturer Symbol Novus Biologicals manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Novus Biologicals mouse anti γh2ax
    a Immunostaining for <t>γH2AX</t> (green), SFTPC (red), DAPI (blue) upper panels; active CASP3 (green), SFTPC (red), AGER (gray), DAPI (blue) middle panels; AGER (green), Sftpc -tdT (red), DAPI (blue) lower panels in lungs of let-7afd AT2 and control mice after 1 month of iTAM. Arrows indicate γH2AX + SFTPC + cells (upper panel) or active-CASP3 + SFTPC + cells (middle panels). Scale bars: 25 μm. b - c Quantification of γH2AX + SFTPC + cells ( b ) or active-CASP3 + SFTPC + cells ( c ) in total SFTPC + cells (n = 3 mice per group). Data are mean±s.e.m. ****p < 0.0001, **p < 0.01, by unpaired Student’s t test. d Representative β-galactosidase staining in lungs of let-7afd AT2 and control mice after 1-month of iTAM. Arrowheads point to AT2 cells in thickened alveolar septa (n = 6 mice per group). Scale bars: 50 μm. e qPCR detection of Trp53 from let-7afd -/- and control Sftpc -tdT + cells after 2 months of iTAM (n = 4 samples per group from pools of 2 mice). Data are mean±s.e.m. *p < 0.05, by unpaired Student’s t test. f Quantification of AGER + Sftpc -tdT + in total Sftpc -tdT + cells from lungs of let-7afd AT2 and control mice at 1-or 5-months after iTAM. Data are mean±s.e.m, by unpaired Student’s t test. All panels are representative of three independent experiments.
    Mouse Anti γh2ax, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti γh2ax/product/Novus Biologicals
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti γh2ax - by Bioz Stars, 2024-07
    94/100 stars

    Images

    1) Product Images from "Let-7 restrains an oncogenic epigenetic circuit in AT2 cells to prevent ectopic formation of fibrogenic transitional cell intermediates and pulmonary fibrosis"

    Article Title: Let-7 restrains an oncogenic epigenetic circuit in AT2 cells to prevent ectopic formation of fibrogenic transitional cell intermediates and pulmonary fibrosis

    Journal: bioRxiv

    doi: 10.1101/2024.05.22.595205

    a Immunostaining for γH2AX (green), SFTPC (red), DAPI (blue) upper panels; active CASP3 (green), SFTPC (red), AGER (gray), DAPI (blue) middle panels; AGER (green), Sftpc -tdT (red), DAPI (blue) lower panels in lungs of let-7afd AT2 and control mice after 1 month of iTAM. Arrows indicate γH2AX + SFTPC + cells (upper panel) or active-CASP3 + SFTPC + cells (middle panels). Scale bars: 25 μm. b - c Quantification of γH2AX + SFTPC + cells ( b ) or active-CASP3 + SFTPC + cells ( c ) in total SFTPC + cells (n = 3 mice per group). Data are mean±s.e.m. ****p < 0.0001, **p < 0.01, by unpaired Student’s t test. d Representative β-galactosidase staining in lungs of let-7afd AT2 and control mice after 1-month of iTAM. Arrowheads point to AT2 cells in thickened alveolar septa (n = 6 mice per group). Scale bars: 50 μm. e qPCR detection of Trp53 from let-7afd -/- and control Sftpc -tdT + cells after 2 months of iTAM (n = 4 samples per group from pools of 2 mice). Data are mean±s.e.m. *p < 0.05, by unpaired Student’s t test. f Quantification of AGER + Sftpc -tdT + in total Sftpc -tdT + cells from lungs of let-7afd AT2 and control mice at 1-or 5-months after iTAM. Data are mean±s.e.m, by unpaired Student’s t test. All panels are representative of three independent experiments.
    Figure Legend Snippet: a Immunostaining for γH2AX (green), SFTPC (red), DAPI (blue) upper panels; active CASP3 (green), SFTPC (red), AGER (gray), DAPI (blue) middle panels; AGER (green), Sftpc -tdT (red), DAPI (blue) lower panels in lungs of let-7afd AT2 and control mice after 1 month of iTAM. Arrows indicate γH2AX + SFTPC + cells (upper panel) or active-CASP3 + SFTPC + cells (middle panels). Scale bars: 25 μm. b - c Quantification of γH2AX + SFTPC + cells ( b ) or active-CASP3 + SFTPC + cells ( c ) in total SFTPC + cells (n = 3 mice per group). Data are mean±s.e.m. ****p < 0.0001, **p < 0.01, by unpaired Student’s t test. d Representative β-galactosidase staining in lungs of let-7afd AT2 and control mice after 1-month of iTAM. Arrowheads point to AT2 cells in thickened alveolar septa (n = 6 mice per group). Scale bars: 50 μm. e qPCR detection of Trp53 from let-7afd -/- and control Sftpc -tdT + cells after 2 months of iTAM (n = 4 samples per group from pools of 2 mice). Data are mean±s.e.m. *p < 0.05, by unpaired Student’s t test. f Quantification of AGER + Sftpc -tdT + in total Sftpc -tdT + cells from lungs of let-7afd AT2 and control mice at 1-or 5-months after iTAM. Data are mean±s.e.m, by unpaired Student’s t test. All panels are representative of three independent experiments.

    Techniques Used: Immunostaining, Staining

    mouse anti γh2ax  (Novus Biologicals)


    Bioz Verified Symbol Novus Biologicals is a verified supplier
    Bioz Manufacturer Symbol Novus Biologicals manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Novus Biologicals mouse anti γh2ax
    a Immunostaining for <t>γH2AX</t> (green), SFTPC (red), DAPI (blue) upper panels; active CASP3 (green), SFTPC (red), AGER (gray), DAPI (blue) middle panels; AGER (green), Sftpc -tdT (red), DAPI (blue) lower panels in lungs of let-7afd AT2 and control mice after 1 month of iTAM. Arrows indicate γH2AX + SFTPC + cells (upper panel) or active-CASP3 + SFTPC + cells (middle panels). Scale bars: 25 μm. b - c Quantification of γH2AX + SFTPC + cells ( b ) or active-CASP3 + SFTPC + cells ( c ) in total SFTPC + cells (n = 3 mice per group). Data are mean±s.e.m. ****p < 0.0001, **p < 0.01, by unpaired Student’s t test. d Representative β-galactosidase staining in lungs of let-7afd AT2 and control mice after 1-month of iTAM. Arrowheads point to AT2 cells in thickened alveolar septa (n = 6 mice per group). Scale bars: 50 μm. e qPCR detection of Trp53 from let-7afd -/- and control Sftpc -tdT + cells after 2 months of iTAM (n = 4 samples per group from pools of 2 mice). Data are mean±s.e.m. *p < 0.05, by unpaired Student’s t test. f Quantification of AGER + Sftpc -tdT + in total Sftpc -tdT + cells from lungs of let-7afd AT2 and control mice at 1-or 5-months after iTAM. Data are mean±s.e.m, by unpaired Student’s t test. All panels are representative of three independent experiments.
    Mouse Anti γh2ax, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti γh2ax/product/Novus Biologicals
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti γh2ax - by Bioz Stars, 2024-07
    94/100 stars

    Images

    1) Product Images from "Let-7 restrains an oncogenic epigenetic circuit in AT2 cells to prevent ectopic formation of fibrogenic transitional cell intermediates and pulmonary fibrosis"

    Article Title: Let-7 restrains an oncogenic epigenetic circuit in AT2 cells to prevent ectopic formation of fibrogenic transitional cell intermediates and pulmonary fibrosis

    Journal: bioRxiv

    doi: 10.1101/2024.05.22.595205

    a Immunostaining for γH2AX (green), SFTPC (red), DAPI (blue) upper panels; active CASP3 (green), SFTPC (red), AGER (gray), DAPI (blue) middle panels; AGER (green), Sftpc -tdT (red), DAPI (blue) lower panels in lungs of let-7afd AT2 and control mice after 1 month of iTAM. Arrows indicate γH2AX + SFTPC + cells (upper panel) or active-CASP3 + SFTPC + cells (middle panels). Scale bars: 25 μm. b - c Quantification of γH2AX + SFTPC + cells ( b ) or active-CASP3 + SFTPC + cells ( c ) in total SFTPC + cells (n = 3 mice per group). Data are mean±s.e.m. ****p < 0.0001, **p < 0.01, by unpaired Student’s t test. d Representative β-galactosidase staining in lungs of let-7afd AT2 and control mice after 1-month of iTAM. Arrowheads point to AT2 cells in thickened alveolar septa (n = 6 mice per group). Scale bars: 50 μm. e qPCR detection of Trp53 from let-7afd -/- and control Sftpc -tdT + cells after 2 months of iTAM (n = 4 samples per group from pools of 2 mice). Data are mean±s.e.m. *p < 0.05, by unpaired Student’s t test. f Quantification of AGER + Sftpc -tdT + in total Sftpc -tdT + cells from lungs of let-7afd AT2 and control mice at 1-or 5-months after iTAM. Data are mean±s.e.m, by unpaired Student’s t test. All panels are representative of three independent experiments.
    Figure Legend Snippet: a Immunostaining for γH2AX (green), SFTPC (red), DAPI (blue) upper panels; active CASP3 (green), SFTPC (red), AGER (gray), DAPI (blue) middle panels; AGER (green), Sftpc -tdT (red), DAPI (blue) lower panels in lungs of let-7afd AT2 and control mice after 1 month of iTAM. Arrows indicate γH2AX + SFTPC + cells (upper panel) or active-CASP3 + SFTPC + cells (middle panels). Scale bars: 25 μm. b - c Quantification of γH2AX + SFTPC + cells ( b ) or active-CASP3 + SFTPC + cells ( c ) in total SFTPC + cells (n = 3 mice per group). Data are mean±s.e.m. ****p < 0.0001, **p < 0.01, by unpaired Student’s t test. d Representative β-galactosidase staining in lungs of let-7afd AT2 and control mice after 1-month of iTAM. Arrowheads point to AT2 cells in thickened alveolar septa (n = 6 mice per group). Scale bars: 50 μm. e qPCR detection of Trp53 from let-7afd -/- and control Sftpc -tdT + cells after 2 months of iTAM (n = 4 samples per group from pools of 2 mice). Data are mean±s.e.m. *p < 0.05, by unpaired Student’s t test. f Quantification of AGER + Sftpc -tdT + in total Sftpc -tdT + cells from lungs of let-7afd AT2 and control mice at 1-or 5-months after iTAM. Data are mean±s.e.m, by unpaired Student’s t test. All panels are representative of three independent experiments.

    Techniques Used: Immunostaining, Staining


    Structured Review

    Millipore mouse anti p histone h2ax ser139
    Mouse Anti P Histone H2ax Ser139, supplied by Millipore, 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/mouse anti p histone h2ax ser139/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti p histone h2ax ser139 - by Bioz Stars, 2024-07
    86/100 stars

    Images


    Structured Review

    Millipore mouse igg1 anti p histone h2ax ser139
    Mouse Igg1 Anti P Histone H2ax Ser139, supplied by Millipore, 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/mouse igg1 anti p histone h2ax ser139/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse igg1 anti p histone h2ax ser139 - by Bioz Stars, 2024-07
    86/100 stars

    Images


    Structured Review

    Santa Cruz Biotechnology mouse anti p h2ax ser139
    Mouse Anti P H2ax Ser139, supplied by Santa Cruz Biotechnology, 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/mouse anti p h2ax ser139/product/Santa Cruz Biotechnology
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti p h2ax ser139 - by Bioz Stars, 2024-07
    95/100 stars

    Images


    Structured Review

    Revvity Signals mouse anti γ h2ax p ser139 antibody
    Mitotic telomere deprotection is suppressed by WRN helicase independently of its catalytic activities. ( a ) Immunoblot of WRN in IMR-90 E6E7 hTERT cells transduced with shWRN or shScramble for 5 days. GAPDH serves as a loading control. ( b ) Representative images of meta-TIF assay from WRN knockdown cells after treatment with 100 ng/ml colcemid. The images show DAPI (blue), <t>γ-H2AX</t> (red), and telomere FISH (green). Scale bar, 10 µm. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci per chromosome spread in indicated conditions. Violin plots illustrate the distribution of all data and averages from three independent experiments (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Kruskal–Wallis followed by Dunn’s test). ( d ) Immunoblot of WRN in IMR-90 E6E7 hTERT cells expressing exogenous WRN RES or Vector. Cells were transduced with shScramble or shWRN for 5 days before analysis. GAPDH serves as a loading control. ( e ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in c (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Kruskal–Wallis followed by Dunn’s test). ( f ) Immunoblot of WRN in cells expressing WRN RES and indicated WRN mutants. Transduced cells were harvested on day 10 post-infection. GAPDH serves as a loading control. ( g ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.
    Mouse Anti γ H2ax P Ser139 Antibody, supplied by Revvity Signals, 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/mouse anti γ h2ax p ser139 antibody/product/Revvity Signals
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti γ h2ax p ser139 antibody - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection"

    Article Title: A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection

    Journal: Scientific Reports

    doi: 10.1038/s41598-023-27598-0

    Mitotic telomere deprotection is suppressed by WRN helicase independently of its catalytic activities. ( a ) Immunoblot of WRN in IMR-90 E6E7 hTERT cells transduced with shWRN or shScramble for 5 days. GAPDH serves as a loading control. ( b ) Representative images of meta-TIF assay from WRN knockdown cells after treatment with 100 ng/ml colcemid. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci per chromosome spread in indicated conditions. Violin plots illustrate the distribution of all data and averages from three independent experiments (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Kruskal–Wallis followed by Dunn’s test). ( d ) Immunoblot of WRN in IMR-90 E6E7 hTERT cells expressing exogenous WRN RES or Vector. Cells were transduced with shScramble or shWRN for 5 days before analysis. GAPDH serves as a loading control. ( e ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in c (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Kruskal–Wallis followed by Dunn’s test). ( f ) Immunoblot of WRN in cells expressing WRN RES and indicated WRN mutants. Transduced cells were harvested on day 10 post-infection. GAPDH serves as a loading control. ( g ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.
    Figure Legend Snippet: Mitotic telomere deprotection is suppressed by WRN helicase independently of its catalytic activities. ( a ) Immunoblot of WRN in IMR-90 E6E7 hTERT cells transduced with shWRN or shScramble for 5 days. GAPDH serves as a loading control. ( b ) Representative images of meta-TIF assay from WRN knockdown cells after treatment with 100 ng/ml colcemid. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci per chromosome spread in indicated conditions. Violin plots illustrate the distribution of all data and averages from three independent experiments (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Kruskal–Wallis followed by Dunn’s test). ( d ) Immunoblot of WRN in IMR-90 E6E7 hTERT cells expressing exogenous WRN RES or Vector. Cells were transduced with shScramble or shWRN for 5 days before analysis. GAPDH serves as a loading control. ( e ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in c (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Kruskal–Wallis followed by Dunn’s test). ( f ) Immunoblot of WRN in cells expressing WRN RES and indicated WRN mutants. Transduced cells were harvested on day 10 post-infection. GAPDH serves as a loading control. ( g ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.

    Techniques Used: Western Blot, Transduction, Expressing, Plasmid Preparation, Infection

    WRN N-terminus coiled-coil region is sufficient to suppress MAD-TIFs. ( a ) Schematic representation of NLS and 4xFLAG tagged WRN fragments and derivative sub-fragments from the N-terminal WRN 2–499 fragment. Exo, exonuclease domain; Coiled, coiled-coil motif; Helicase, helicase domain; RQC, RecQ C-terminal DNA-binding domain; HRDC, helicase and RNaseD C-terminal domain. ( b ) Immunoblot of endogenous WRN and NLS-4FL-WRN fragments in IMR-90 E6E7 hTERT cells expressing indicated WRN fragments. Transduced cells were analyzed on day 10 post-infection. A black arrowhead indicates bands of the expected size of 60–70 kDa. GAPDH serves as a loading control. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). ( d ) Immunoblot of FLAG-WRN fragments in IMR-90 E6E7 hTERT cells expressing indicated WRN fragments. Transduced cells were harvested on day 12 post-infection. A black arrowhead indicates the expected fragment size (~ 27 kDa). Potential truncation, complex formation, and post-translational modifications are specified with white arrowheads. Asterisks represent unspecific bands from empty Vector. GAPDH serves as a loading control. ( e ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). ( f ) Immunoblot of FLAG-WRN fragments in IMR-90 E6E7 hTERT cells expressing indicated WRN fragments. Transduced cells were harvested on day 12 post-infection. Magenta and blue arrowheads indicate the expected size for the WRN168-333 (~ 27 kDa) and the WRN251-333 (~ 17 kDa) fragments, respectively. White arrowheads with colored lines indicate possible post-translational modifications or complex formation for each mutant. ( g ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.
    Figure Legend Snippet: WRN N-terminus coiled-coil region is sufficient to suppress MAD-TIFs. ( a ) Schematic representation of NLS and 4xFLAG tagged WRN fragments and derivative sub-fragments from the N-terminal WRN 2–499 fragment. Exo, exonuclease domain; Coiled, coiled-coil motif; Helicase, helicase domain; RQC, RecQ C-terminal DNA-binding domain; HRDC, helicase and RNaseD C-terminal domain. ( b ) Immunoblot of endogenous WRN and NLS-4FL-WRN fragments in IMR-90 E6E7 hTERT cells expressing indicated WRN fragments. Transduced cells were analyzed on day 10 post-infection. A black arrowhead indicates bands of the expected size of 60–70 kDa. GAPDH serves as a loading control. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). ( d ) Immunoblot of FLAG-WRN fragments in IMR-90 E6E7 hTERT cells expressing indicated WRN fragments. Transduced cells were harvested on day 12 post-infection. A black arrowhead indicates the expected fragment size (~ 27 kDa). Potential truncation, complex formation, and post-translational modifications are specified with white arrowheads. Asterisks represent unspecific bands from empty Vector. GAPDH serves as a loading control. ( e ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). ( f ) Immunoblot of FLAG-WRN fragments in IMR-90 E6E7 hTERT cells expressing indicated WRN fragments. Transduced cells were harvested on day 12 post-infection. Magenta and blue arrowheads indicate the expected size for the WRN168-333 (~ 27 kDa) and the WRN251-333 (~ 17 kDa) fragments, respectively. White arrowheads with colored lines indicate possible post-translational modifications or complex formation for each mutant. ( g ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.

    Techniques Used: Binding Assay, Western Blot, Expressing, Infection, Plasmid Preparation, Mutagenesis

    WRN N-terminus does not perturb mitotic Aurora B or ATM kinase activities. ( a ) Distribution of mitotic duration in cells expressing indicated WRN fragments (Median, 25th, and 75th percentile; Kruskal–Wallis followed by Dunn's test). Cells were exposed to 500 nM taxol and analyzed by live-cell imaging data. Vector cells were co-treated with 40 nM Hesperadin as a control. ( b ) Ratio of each cell fate after mitosis in indicated cells. Results are separately shown as categorized by the duration of mitotic arrest. Mitosis longer than 2 h was defined as mitotic arrest. ( c ) Schematic of the timing of 100 ng/ml colcemid and 0.2 µg/ml bleomycin treatment in IMR-90 E6E7 hTERT cells expressing NLS-4xFL-WRN 2–499 fragment. ( d ) Representative images of γ-H2AX foci on mitotic chromosomes in cells treated as in ( c ). Images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( e ) Quantification of total γ-H2AX foci on mitotic chromosomes. Violin plots illustrate the distribution of all data and averages from three independent experiments (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test).
    Figure Legend Snippet: WRN N-terminus does not perturb mitotic Aurora B or ATM kinase activities. ( a ) Distribution of mitotic duration in cells expressing indicated WRN fragments (Median, 25th, and 75th percentile; Kruskal–Wallis followed by Dunn's test). Cells were exposed to 500 nM taxol and analyzed by live-cell imaging data. Vector cells were co-treated with 40 nM Hesperadin as a control. ( b ) Ratio of each cell fate after mitosis in indicated cells. Results are separately shown as categorized by the duration of mitotic arrest. Mitosis longer than 2 h was defined as mitotic arrest. ( c ) Schematic of the timing of 100 ng/ml colcemid and 0.2 µg/ml bleomycin treatment in IMR-90 E6E7 hTERT cells expressing NLS-4xFL-WRN 2–499 fragment. ( d ) Representative images of γ-H2AX foci on mitotic chromosomes in cells treated as in ( c ). Images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( e ) Quantification of total γ-H2AX foci on mitotic chromosomes. Violin plots illustrate the distribution of all data and averages from three independent experiments (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test).

    Techniques Used: Expressing, Live Cell Imaging, Plasmid Preparation

    WRN supports TRF2 function to protect mitotic telomeres. ( a ) Immunoblot of endogenous WRN, FLAG-WRN fragments, and TRF2 upon TRF2 depletion in indicated cells. IMR-90 E6E7 hTERT cells expressing indicated WRN fragments were transduced with shTRF2 lentivirus and analyzed on day 7 post-infection. Blue-colored arrowhead indicates the expected size for WRN 168–333 (~ 27 kDa). White arrowheads (magenta line border for WRN 2–499 fragment and blue border for WRN 168–333 ) indicate possible post-translational modifications or complex formation. GAPDH serves as a loading control. ( b ) Representative images of meta-TIF assay from TRF2 knockdown cells expressing indicated WRN fragments after treatment with 100 ng/ml colcemid. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Mann–Whitney test). Dashed lines discriminate between the average number of TIFs generated in the interphase due to shTRF2 (2 h colcemid) and MAD-TIFs caused by mitotic arrest (24 h colcemid). ( d ) Immunoblot of endogenous WRN and TRF2 in indicated cells. IMR-90 E6E7 hTERT cells expressing exogenous TRF2 were transduced with shWRN lentivirus and analyzed on day 5 post-infection. GAPDH serves as a loading control. ( e ) Representative images of meta-TIF assay in indicated cells from d after treatment with 100 ng/ml colcemid for 24 h. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( f ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.
    Figure Legend Snippet: WRN supports TRF2 function to protect mitotic telomeres. ( a ) Immunoblot of endogenous WRN, FLAG-WRN fragments, and TRF2 upon TRF2 depletion in indicated cells. IMR-90 E6E7 hTERT cells expressing indicated WRN fragments were transduced with shTRF2 lentivirus and analyzed on day 7 post-infection. Blue-colored arrowhead indicates the expected size for WRN 168–333 (~ 27 kDa). White arrowheads (magenta line border for WRN 2–499 fragment and blue border for WRN 168–333 ) indicate possible post-translational modifications or complex formation. GAPDH serves as a loading control. ( b ) Representative images of meta-TIF assay from TRF2 knockdown cells expressing indicated WRN fragments after treatment with 100 ng/ml colcemid. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Mann–Whitney test). Dashed lines discriminate between the average number of TIFs generated in the interphase due to shTRF2 (2 h colcemid) and MAD-TIFs caused by mitotic arrest (24 h colcemid). ( d ) Immunoblot of endogenous WRN and TRF2 in indicated cells. IMR-90 E6E7 hTERT cells expressing exogenous TRF2 were transduced with shWRN lentivirus and analyzed on day 5 post-infection. GAPDH serves as a loading control. ( e ) Representative images of meta-TIF assay in indicated cells from d after treatment with 100 ng/ml colcemid for 24 h. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( f ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.

    Techniques Used: Western Blot, Expressing, Transduction, Infection, MANN-WHITNEY, Generated

    Phosphomimetic mutation at S282 of WRN 168–333 disrupts its MAD-TIF suppressor effect. ( a ) Schematic representation of four potential Aurora B sites in the 168–333 aa of WRN N-terminus. Sites of alanine and phosphomimetic mutations are indicated below. All mutants contain N-terminal NLS and 4xFLAG tags. ( b ) Immunoblot of FLAG-WRN fragments in indicated cells. Transduced cells were harvested on day 12 post-infection. A black arrowhead indicates the expected band size for all the mutants (~ 27 kDa), and white arrowheads indicate additional bands. ( c ) Representative images of meta-TIF assay in indicated cells after treatment with 100 ng/ml colcemid for 24 h. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( d ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.
    Figure Legend Snippet: Phosphomimetic mutation at S282 of WRN 168–333 disrupts its MAD-TIF suppressor effect. ( a ) Schematic representation of four potential Aurora B sites in the 168–333 aa of WRN N-terminus. Sites of alanine and phosphomimetic mutations are indicated below. All mutants contain N-terminal NLS and 4xFLAG tags. ( b ) Immunoblot of FLAG-WRN fragments in indicated cells. Transduced cells were harvested on day 12 post-infection. A black arrowhead indicates the expected band size for all the mutants (~ 27 kDa), and white arrowheads indicate additional bands. ( c ) Representative images of meta-TIF assay in indicated cells after treatment with 100 ng/ml colcemid for 24 h. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( d ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.

    Techniques Used: Mutagenesis, Western Blot, Infection

    mouse monoclonal anti γ h2ax p ser139 antibody  (Abcam)

     
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Abcam mouse monoclonal anti γ h2ax p ser139 antibody
    Down-regulation of CK2 results in persistent <t>γ-H2AX</t> signal and reduced colony formation in human M059K glioblastoma cells . A . Cells were transfected with scramble siRNA (si-Scr), CK2α or -α'-siRNA for 72 hours. Where indicated, 0.5 μg/ml neocarzinostatin (NCS) was added to the medium in the last 24 hours of incubation. Fixed cells were subsequently labeled with anti-γ-H2AX antibody and with a FITC-conjugated secondary antibody. Nuclei were visualized by DAPI staining. B . Quantification of γ-H2AX-positive cells was performed by using ImageJ software and expressed as percentage of the total number of cells in each sample. Bars indicate mean values +/- standard deviation (SD) from three independent experiments. *P < 0.0001 indicates statistically significant difference in the number of γ-H2AX-positive cells in CK2-depleted versus si-Scr-treated cells. C . Cells were transfected with CK2α-, -α'-siRNA or scramble siRNA for 72 hours. 0.5 μg/ml NCS was added in the last hour of incubation. Control, refers to cell treated with transfection reagent only. Cells were allowed to form clusters for 14 days. Colonies were visualized by staining with crystal violet as described in Experimental Procedures. Bar graph shows cell colonies quantification. Average values +/- SD from three independent experiments are shown relative to control (i.e. transfection reagent-treated) cells. *P < 0.005 denotes statistically significant difference in number of colonies formed as compared to si-Scr-tranfected and NCS treated cells.
    Mouse Monoclonal Anti γ H2ax P Ser139 Antibody, supplied by Abcam, 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/mouse monoclonal anti γ h2ax p ser139 antibody/product/Abcam
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti γ h2ax p ser139 antibody - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage"

    Article Title: Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-13-7

    Down-regulation of CK2 results in persistent γ-H2AX signal and reduced colony formation in human M059K glioblastoma cells . A . Cells were transfected with scramble siRNA (si-Scr), CK2α or -α'-siRNA for 72 hours. Where indicated, 0.5 μg/ml neocarzinostatin (NCS) was added to the medium in the last 24 hours of incubation. Fixed cells were subsequently labeled with anti-γ-H2AX antibody and with a FITC-conjugated secondary antibody. Nuclei were visualized by DAPI staining. B . Quantification of γ-H2AX-positive cells was performed by using ImageJ software and expressed as percentage of the total number of cells in each sample. Bars indicate mean values +/- standard deviation (SD) from three independent experiments. *P < 0.0001 indicates statistically significant difference in the number of γ-H2AX-positive cells in CK2-depleted versus si-Scr-treated cells. C . Cells were transfected with CK2α-, -α'-siRNA or scramble siRNA for 72 hours. 0.5 μg/ml NCS was added in the last hour of incubation. Control, refers to cell treated with transfection reagent only. Cells were allowed to form clusters for 14 days. Colonies were visualized by staining with crystal violet as described in Experimental Procedures. Bar graph shows cell colonies quantification. Average values +/- SD from three independent experiments are shown relative to control (i.e. transfection reagent-treated) cells. *P < 0.005 denotes statistically significant difference in number of colonies formed as compared to si-Scr-tranfected and NCS treated cells.
    Figure Legend Snippet: Down-regulation of CK2 results in persistent γ-H2AX signal and reduced colony formation in human M059K glioblastoma cells . A . Cells were transfected with scramble siRNA (si-Scr), CK2α or -α'-siRNA for 72 hours. Where indicated, 0.5 μg/ml neocarzinostatin (NCS) was added to the medium in the last 24 hours of incubation. Fixed cells were subsequently labeled with anti-γ-H2AX antibody and with a FITC-conjugated secondary antibody. Nuclei were visualized by DAPI staining. B . Quantification of γ-H2AX-positive cells was performed by using ImageJ software and expressed as percentage of the total number of cells in each sample. Bars indicate mean values +/- standard deviation (SD) from three independent experiments. *P < 0.0001 indicates statistically significant difference in the number of γ-H2AX-positive cells in CK2-depleted versus si-Scr-treated cells. C . Cells were transfected with CK2α-, -α'-siRNA or scramble siRNA for 72 hours. 0.5 μg/ml NCS was added in the last hour of incubation. Control, refers to cell treated with transfection reagent only. Cells were allowed to form clusters for 14 days. Colonies were visualized by staining with crystal violet as described in Experimental Procedures. Bar graph shows cell colonies quantification. Average values +/- SD from three independent experiments are shown relative to control (i.e. transfection reagent-treated) cells. *P < 0.005 denotes statistically significant difference in number of colonies formed as compared to si-Scr-tranfected and NCS treated cells.

    Techniques Used: Transfection, Incubation, Labeling, Staining, Software, Standard Deviation

    CK2 co-localizes with γ-H2AX to sites of DNA damage . A . Association between CK2α' and γ-H2AX was revealed by in situ PLA. Cells expressing or lacking CK2α' were left untreated or incubated with 0.5 μg/ml NCS for 1 hour before fixation and labeling with primary antibodies directed against the indicated proteins. Control, refers to cells transfected with si-CK2α' and stained with secondary antibodies. Cell nuclei were revealed by Hoechst staining. B . Quantification of the number of signals/cell as distinct fluorescent red spots was performed by computer-assisted image analysis as described in Experimental Procedures. Mean values +/- SD from three independent experiments are shown. *P < 0.0001 denotes statistically significant difference.
    Figure Legend Snippet: CK2 co-localizes with γ-H2AX to sites of DNA damage . A . Association between CK2α' and γ-H2AX was revealed by in situ PLA. Cells expressing or lacking CK2α' were left untreated or incubated with 0.5 μg/ml NCS for 1 hour before fixation and labeling with primary antibodies directed against the indicated proteins. Control, refers to cells transfected with si-CK2α' and stained with secondary antibodies. Cell nuclei were revealed by Hoechst staining. B . Quantification of the number of signals/cell as distinct fluorescent red spots was performed by computer-assisted image analysis as described in Experimental Procedures. Mean values +/- SD from three independent experiments are shown. *P < 0.0001 denotes statistically significant difference.

    Techniques Used: In Situ, Expressing, Incubation, Labeling, Transfection, Staining

    Cells overexpressing CK2 exhibit a prompt response to induction of DNA double-strand break . A . M059K cells transiently overexpressing CK2α'-DsRed or DsRed-fluorescent protein were incubated with 0.5 μg/ml NCS for the indicated times. Subsequently, cells were fixed and labeled with anti-γ-H2AX antibody. Cell nuclei were revealed by DAPI staining. Arrows: cells overexpressing CK2α'-DsRed are more efficient in DNA damage repair as compared to cells that do not overexpress the kinase. One representative experiment is shown. B . Average γ-H2AX foci number per red fluorescent cell as described in A . Mean values +/- SD from three independent experiments are shown. More than 100 cells have been analyzed per experiment. *P < 0.001 denotes statistically significant difference. NS, not significant.
    Figure Legend Snippet: Cells overexpressing CK2 exhibit a prompt response to induction of DNA double-strand break . A . M059K cells transiently overexpressing CK2α'-DsRed or DsRed-fluorescent protein were incubated with 0.5 μg/ml NCS for the indicated times. Subsequently, cells were fixed and labeled with anti-γ-H2AX antibody. Cell nuclei were revealed by DAPI staining. Arrows: cells overexpressing CK2α'-DsRed are more efficient in DNA damage repair as compared to cells that do not overexpress the kinase. One representative experiment is shown. B . Average γ-H2AX foci number per red fluorescent cell as described in A . Mean values +/- SD from three independent experiments are shown. More than 100 cells have been analyzed per experiment. *P < 0.001 denotes statistically significant difference. NS, not significant.

    Techniques Used: Incubation, Labeling, Staining

    mouse p h2ax ser139  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Cell Signaling Technology Inc mouse p h2ax ser139
    Mouse P H2ax Ser139, 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/mouse p h2ax ser139/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse p h2ax ser139 - by Bioz Stars, 2024-07
    86/100 stars

    Images


    Structured Review

    Millipore mouse p h2ax ser139
    Mouse P H2ax Ser139, supplied by Millipore, 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/mouse p h2ax ser139/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse p h2ax ser139 - by Bioz Stars, 2024-07
    86/100 stars

    Images

    p h2ax  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    Cell Signaling Technology Inc p h2ax
    S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of <t>p-H2ax</t> in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.
    P H2ax, 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/p h2ax/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p h2ax - by Bioz Stars, 2024-07
    95/100 stars

    Images

    1) Product Images from "Sphingosine-1-Phosphate Alleviates Irradiation Induced Salivary Gland Hypofunction through Preserving Endothelial Cells and Resident Macrophages"

    Article Title: Sphingosine-1-Phosphate Alleviates Irradiation Induced Salivary Gland Hypofunction through Preserving Endothelial Cells and Resident Macrophages

    Journal: Antioxidants

    doi: 10.3390/antiox11102050

    S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of p-H2ax in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.
    Figure Legend Snippet: S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of p-H2ax in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

    Techniques Used: Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

    Effects of S1P on endothelial cells and macrophages in radiated salivary glands. ( A – D ) Expression of S1pr1 and Cd31 in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( E , F ) Levels of p-H2ax in Cd31 + endothelial cells in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( G – I ) Expression of F4/80 and levels of p-H2ax in F4/80 + macrophages in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( J ) mRNA levels of macrophage-enriched Hgf and endothelia-enriched Kitl in SMGs collected at 7 days after IR were examined with RT-qPCR. Quantified data are shown as mean ± SD, N = 3, NS: not significant vs. NT, ND: not detected, # : p < 0.05 vs. NT, *: p < 0.05, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.
    Figure Legend Snippet: Effects of S1P on endothelial cells and macrophages in radiated salivary glands. ( A – D ) Expression of S1pr1 and Cd31 in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( E , F ) Levels of p-H2ax in Cd31 + endothelial cells in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( G – I ) Expression of F4/80 and levels of p-H2ax in F4/80 + macrophages in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( J ) mRNA levels of macrophage-enriched Hgf and endothelia-enriched Kitl in SMGs collected at 7 days after IR were examined with RT-qPCR. Quantified data are shown as mean ± SD, N = 3, NS: not significant vs. NT, ND: not detected, # : p < 0.05 vs. NT, *: p < 0.05, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

    Techniques Used: Expressing, Double Immunofluorescence Staining, Quantitative RT-PCR

    anti p h2ax  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    Cell Signaling Technology Inc anti p h2ax
    S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of <t>p-H2ax</t> in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.
    Anti P H2ax, 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/anti p h2ax/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti p h2ax - by Bioz Stars, 2024-07
    95/100 stars

    Images

    1) Product Images from "Sphingosine-1-Phosphate Alleviates Irradiation Induced Salivary Gland Hypofunction through Preserving Endothelial Cells and Resident Macrophages"

    Article Title: Sphingosine-1-Phosphate Alleviates Irradiation Induced Salivary Gland Hypofunction through Preserving Endothelial Cells and Resident Macrophages

    Journal: Antioxidants

    doi: 10.3390/antiox11102050

    S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of p-H2ax in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.
    Figure Legend Snippet: S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of p-H2ax in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

    Techniques Used: Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

    Effects of S1P on endothelial cells and macrophages in radiated salivary glands. ( A – D ) Expression of S1pr1 and Cd31 in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( E , F ) Levels of p-H2ax in Cd31 + endothelial cells in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( G – I ) Expression of F4/80 and levels of p-H2ax in F4/80 + macrophages in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( J ) mRNA levels of macrophage-enriched Hgf and endothelia-enriched Kitl in SMGs collected at 7 days after IR were examined with RT-qPCR. Quantified data are shown as mean ± SD, N = 3, NS: not significant vs. NT, ND: not detected, # : p < 0.05 vs. NT, *: p < 0.05, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.
    Figure Legend Snippet: Effects of S1P on endothelial cells and macrophages in radiated salivary glands. ( A – D ) Expression of S1pr1 and Cd31 in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( E , F ) Levels of p-H2ax in Cd31 + endothelial cells in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( G – I ) Expression of F4/80 and levels of p-H2ax in F4/80 + macrophages in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( J ) mRNA levels of macrophage-enriched Hgf and endothelia-enriched Kitl in SMGs collected at 7 days after IR were examined with RT-qPCR. Quantified data are shown as mean ± SD, N = 3, NS: not significant vs. NT, ND: not detected, # : p < 0.05 vs. NT, *: p < 0.05, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

    Techniques Used: Expressing, Double Immunofluorescence Staining, Quantitative RT-PCR

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Novus Biologicals mouse anti γh2ax
    a Immunostaining for <t>γH2AX</t> (green), SFTPC (red), DAPI (blue) upper panels; active CASP3 (green), SFTPC (red), AGER (gray), DAPI (blue) middle panels; AGER (green), Sftpc -tdT (red), DAPI (blue) lower panels in lungs of let-7afd AT2 and control mice after 1 month of iTAM. Arrows indicate γH2AX + SFTPC + cells (upper panel) or active-CASP3 + SFTPC + cells (middle panels). Scale bars: 25 μm. b - c Quantification of γH2AX + SFTPC + cells ( b ) or active-CASP3 + SFTPC + cells ( c ) in total SFTPC + cells (n = 3 mice per group). Data are mean±s.e.m. ****p < 0.0001, **p < 0.01, by unpaired Student’s t test. d Representative β-galactosidase staining in lungs of let-7afd AT2 and control mice after 1-month of iTAM. Arrowheads point to AT2 cells in thickened alveolar septa (n = 6 mice per group). Scale bars: 50 μm. e qPCR detection of Trp53 from let-7afd -/- and control Sftpc -tdT + cells after 2 months of iTAM (n = 4 samples per group from pools of 2 mice). Data are mean±s.e.m. *p < 0.05, by unpaired Student’s t test. f Quantification of AGER + Sftpc -tdT + in total Sftpc -tdT + cells from lungs of let-7afd AT2 and control mice at 1-or 5-months after iTAM. Data are mean±s.e.m, by unpaired Student’s t test. All panels are representative of three independent experiments.
    Mouse Anti γh2ax, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti γh2ax/product/Novus Biologicals
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti γh2ax - by Bioz Stars, 2024-07
    94/100 stars
      Buy from Supplier

    86
    Millipore mouse anti p histone h2ax ser139
    a Immunostaining for <t>γH2AX</t> (green), SFTPC (red), DAPI (blue) upper panels; active CASP3 (green), SFTPC (red), AGER (gray), DAPI (blue) middle panels; AGER (green), Sftpc -tdT (red), DAPI (blue) lower panels in lungs of let-7afd AT2 and control mice after 1 month of iTAM. Arrows indicate γH2AX + SFTPC + cells (upper panel) or active-CASP3 + SFTPC + cells (middle panels). Scale bars: 25 μm. b - c Quantification of γH2AX + SFTPC + cells ( b ) or active-CASP3 + SFTPC + cells ( c ) in total SFTPC + cells (n = 3 mice per group). Data are mean±s.e.m. ****p < 0.0001, **p < 0.01, by unpaired Student’s t test. d Representative β-galactosidase staining in lungs of let-7afd AT2 and control mice after 1-month of iTAM. Arrowheads point to AT2 cells in thickened alveolar septa (n = 6 mice per group). Scale bars: 50 μm. e qPCR detection of Trp53 from let-7afd -/- and control Sftpc -tdT + cells after 2 months of iTAM (n = 4 samples per group from pools of 2 mice). Data are mean±s.e.m. *p < 0.05, by unpaired Student’s t test. f Quantification of AGER + Sftpc -tdT + in total Sftpc -tdT + cells from lungs of let-7afd AT2 and control mice at 1-or 5-months after iTAM. Data are mean±s.e.m, by unpaired Student’s t test. All panels are representative of three independent experiments.
    Mouse Anti P Histone H2ax Ser139, supplied by Millipore, 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/mouse anti p histone h2ax ser139/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti p histone h2ax ser139 - by Bioz Stars, 2024-07
    86/100 stars
      Buy from Supplier

    86
    Millipore mouse igg1 anti p histone h2ax ser139
    a Immunostaining for <t>γH2AX</t> (green), SFTPC (red), DAPI (blue) upper panels; active CASP3 (green), SFTPC (red), AGER (gray), DAPI (blue) middle panels; AGER (green), Sftpc -tdT (red), DAPI (blue) lower panels in lungs of let-7afd AT2 and control mice after 1 month of iTAM. Arrows indicate γH2AX + SFTPC + cells (upper panel) or active-CASP3 + SFTPC + cells (middle panels). Scale bars: 25 μm. b - c Quantification of γH2AX + SFTPC + cells ( b ) or active-CASP3 + SFTPC + cells ( c ) in total SFTPC + cells (n = 3 mice per group). Data are mean±s.e.m. ****p < 0.0001, **p < 0.01, by unpaired Student’s t test. d Representative β-galactosidase staining in lungs of let-7afd AT2 and control mice after 1-month of iTAM. Arrowheads point to AT2 cells in thickened alveolar septa (n = 6 mice per group). Scale bars: 50 μm. e qPCR detection of Trp53 from let-7afd -/- and control Sftpc -tdT + cells after 2 months of iTAM (n = 4 samples per group from pools of 2 mice). Data are mean±s.e.m. *p < 0.05, by unpaired Student’s t test. f Quantification of AGER + Sftpc -tdT + in total Sftpc -tdT + cells from lungs of let-7afd AT2 and control mice at 1-or 5-months after iTAM. Data are mean±s.e.m, by unpaired Student’s t test. All panels are representative of three independent experiments.
    Mouse Igg1 Anti P Histone H2ax Ser139, supplied by Millipore, 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/mouse igg1 anti p histone h2ax ser139/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse igg1 anti p histone h2ax ser139 - by Bioz Stars, 2024-07
    86/100 stars
      Buy from Supplier

    95
    Santa Cruz Biotechnology mouse anti p h2ax ser139
    a Immunostaining for <t>γH2AX</t> (green), SFTPC (red), DAPI (blue) upper panels; active CASP3 (green), SFTPC (red), AGER (gray), DAPI (blue) middle panels; AGER (green), Sftpc -tdT (red), DAPI (blue) lower panels in lungs of let-7afd AT2 and control mice after 1 month of iTAM. Arrows indicate γH2AX + SFTPC + cells (upper panel) or active-CASP3 + SFTPC + cells (middle panels). Scale bars: 25 μm. b - c Quantification of γH2AX + SFTPC + cells ( b ) or active-CASP3 + SFTPC + cells ( c ) in total SFTPC + cells (n = 3 mice per group). Data are mean±s.e.m. ****p < 0.0001, **p < 0.01, by unpaired Student’s t test. d Representative β-galactosidase staining in lungs of let-7afd AT2 and control mice after 1-month of iTAM. Arrowheads point to AT2 cells in thickened alveolar septa (n = 6 mice per group). Scale bars: 50 μm. e qPCR detection of Trp53 from let-7afd -/- and control Sftpc -tdT + cells after 2 months of iTAM (n = 4 samples per group from pools of 2 mice). Data are mean±s.e.m. *p < 0.05, by unpaired Student’s t test. f Quantification of AGER + Sftpc -tdT + in total Sftpc -tdT + cells from lungs of let-7afd AT2 and control mice at 1-or 5-months after iTAM. Data are mean±s.e.m, by unpaired Student’s t test. All panels are representative of three independent experiments.
    Mouse Anti P H2ax Ser139, supplied by Santa Cruz Biotechnology, 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/mouse anti p h2ax ser139/product/Santa Cruz Biotechnology
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti p h2ax ser139 - by Bioz Stars, 2024-07
    95/100 stars
      Buy from Supplier

    86
    Revvity Signals mouse anti γ h2ax p ser139 antibody
    Mitotic telomere deprotection is suppressed by WRN helicase independently of its catalytic activities. ( a ) Immunoblot of WRN in IMR-90 E6E7 hTERT cells transduced with shWRN or shScramble for 5 days. GAPDH serves as a loading control. ( b ) Representative images of meta-TIF assay from WRN knockdown cells after treatment with 100 ng/ml colcemid. The images show DAPI (blue), <t>γ-H2AX</t> (red), and telomere FISH (green). Scale bar, 10 µm. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci per chromosome spread in indicated conditions. Violin plots illustrate the distribution of all data and averages from three independent experiments (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Kruskal–Wallis followed by Dunn’s test). ( d ) Immunoblot of WRN in IMR-90 E6E7 hTERT cells expressing exogenous WRN RES or Vector. Cells were transduced with shScramble or shWRN for 5 days before analysis. GAPDH serves as a loading control. ( e ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in c (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Kruskal–Wallis followed by Dunn’s test). ( f ) Immunoblot of WRN in cells expressing WRN RES and indicated WRN mutants. Transduced cells were harvested on day 10 post-infection. GAPDH serves as a loading control. ( g ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.
    Mouse Anti γ H2ax P Ser139 Antibody, supplied by Revvity Signals, 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/mouse anti γ h2ax p ser139 antibody/product/Revvity Signals
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti γ h2ax p ser139 antibody - by Bioz Stars, 2024-07
    86/100 stars
      Buy from Supplier

    86
    Abcam mouse monoclonal anti γ h2ax p ser139 antibody
    Down-regulation of CK2 results in persistent <t>γ-H2AX</t> signal and reduced colony formation in human M059K glioblastoma cells . A . Cells were transfected with scramble siRNA (si-Scr), CK2α or -α'-siRNA for 72 hours. Where indicated, 0.5 μg/ml neocarzinostatin (NCS) was added to the medium in the last 24 hours of incubation. Fixed cells were subsequently labeled with anti-γ-H2AX antibody and with a FITC-conjugated secondary antibody. Nuclei were visualized by DAPI staining. B . Quantification of γ-H2AX-positive cells was performed by using ImageJ software and expressed as percentage of the total number of cells in each sample. Bars indicate mean values +/- standard deviation (SD) from three independent experiments. *P < 0.0001 indicates statistically significant difference in the number of γ-H2AX-positive cells in CK2-depleted versus si-Scr-treated cells. C . Cells were transfected with CK2α-, -α'-siRNA or scramble siRNA for 72 hours. 0.5 μg/ml NCS was added in the last hour of incubation. Control, refers to cell treated with transfection reagent only. Cells were allowed to form clusters for 14 days. Colonies were visualized by staining with crystal violet as described in Experimental Procedures. Bar graph shows cell colonies quantification. Average values +/- SD from three independent experiments are shown relative to control (i.e. transfection reagent-treated) cells. *P < 0.005 denotes statistically significant difference in number of colonies formed as compared to si-Scr-tranfected and NCS treated cells.
    Mouse Monoclonal Anti γ H2ax P Ser139 Antibody, supplied by Abcam, 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/mouse monoclonal anti γ h2ax p ser139 antibody/product/Abcam
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse monoclonal anti γ h2ax p ser139 antibody - by Bioz Stars, 2024-07
    86/100 stars
      Buy from Supplier

    86
    Cell Signaling Technology Inc mouse p h2ax ser139
    Down-regulation of CK2 results in persistent <t>γ-H2AX</t> signal and reduced colony formation in human M059K glioblastoma cells . A . Cells were transfected with scramble siRNA (si-Scr), CK2α or -α'-siRNA for 72 hours. Where indicated, 0.5 μg/ml neocarzinostatin (NCS) was added to the medium in the last 24 hours of incubation. Fixed cells were subsequently labeled with anti-γ-H2AX antibody and with a FITC-conjugated secondary antibody. Nuclei were visualized by DAPI staining. B . Quantification of γ-H2AX-positive cells was performed by using ImageJ software and expressed as percentage of the total number of cells in each sample. Bars indicate mean values +/- standard deviation (SD) from three independent experiments. *P < 0.0001 indicates statistically significant difference in the number of γ-H2AX-positive cells in CK2-depleted versus si-Scr-treated cells. C . Cells were transfected with CK2α-, -α'-siRNA or scramble siRNA for 72 hours. 0.5 μg/ml NCS was added in the last hour of incubation. Control, refers to cell treated with transfection reagent only. Cells were allowed to form clusters for 14 days. Colonies were visualized by staining with crystal violet as described in Experimental Procedures. Bar graph shows cell colonies quantification. Average values +/- SD from three independent experiments are shown relative to control (i.e. transfection reagent-treated) cells. *P < 0.005 denotes statistically significant difference in number of colonies formed as compared to si-Scr-tranfected and NCS treated cells.
    Mouse P H2ax Ser139, 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/mouse p h2ax ser139/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse p h2ax ser139 - by Bioz Stars, 2024-07
    86/100 stars
      Buy from Supplier

    86
    Millipore mouse p h2ax ser139
    Down-regulation of CK2 results in persistent <t>γ-H2AX</t> signal and reduced colony formation in human M059K glioblastoma cells . A . Cells were transfected with scramble siRNA (si-Scr), CK2α or -α'-siRNA for 72 hours. Where indicated, 0.5 μg/ml neocarzinostatin (NCS) was added to the medium in the last 24 hours of incubation. Fixed cells were subsequently labeled with anti-γ-H2AX antibody and with a FITC-conjugated secondary antibody. Nuclei were visualized by DAPI staining. B . Quantification of γ-H2AX-positive cells was performed by using ImageJ software and expressed as percentage of the total number of cells in each sample. Bars indicate mean values +/- standard deviation (SD) from three independent experiments. *P < 0.0001 indicates statistically significant difference in the number of γ-H2AX-positive cells in CK2-depleted versus si-Scr-treated cells. C . Cells were transfected with CK2α-, -α'-siRNA or scramble siRNA for 72 hours. 0.5 μg/ml NCS was added in the last hour of incubation. Control, refers to cell treated with transfection reagent only. Cells were allowed to form clusters for 14 days. Colonies were visualized by staining with crystal violet as described in Experimental Procedures. Bar graph shows cell colonies quantification. Average values +/- SD from three independent experiments are shown relative to control (i.e. transfection reagent-treated) cells. *P < 0.005 denotes statistically significant difference in number of colonies formed as compared to si-Scr-tranfected and NCS treated cells.
    Mouse P H2ax Ser139, supplied by Millipore, 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/mouse p h2ax ser139/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse p h2ax ser139 - by Bioz Stars, 2024-07
    86/100 stars
      Buy from Supplier

    95
    Cell Signaling Technology Inc p h2ax
    S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of <t>p-H2ax</t> in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.
    P H2ax, 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/p h2ax/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p h2ax - by Bioz Stars, 2024-07
    95/100 stars
      Buy from Supplier

    95
    Cell Signaling Technology Inc anti p h2ax
    S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of <t>p-H2ax</t> in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.
    Anti P H2ax, 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/anti p h2ax/product/Cell Signaling Technology Inc
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti p h2ax - by Bioz Stars, 2024-07
    95/100 stars
      Buy from Supplier

    Image Search Results


    a Immunostaining for γH2AX (green), SFTPC (red), DAPI (blue) upper panels; active CASP3 (green), SFTPC (red), AGER (gray), DAPI (blue) middle panels; AGER (green), Sftpc -tdT (red), DAPI (blue) lower panels in lungs of let-7afd AT2 and control mice after 1 month of iTAM. Arrows indicate γH2AX + SFTPC + cells (upper panel) or active-CASP3 + SFTPC + cells (middle panels). Scale bars: 25 μm. b - c Quantification of γH2AX + SFTPC + cells ( b ) or active-CASP3 + SFTPC + cells ( c ) in total SFTPC + cells (n = 3 mice per group). Data are mean±s.e.m. ****p < 0.0001, **p < 0.01, by unpaired Student’s t test. d Representative β-galactosidase staining in lungs of let-7afd AT2 and control mice after 1-month of iTAM. Arrowheads point to AT2 cells in thickened alveolar septa (n = 6 mice per group). Scale bars: 50 μm. e qPCR detection of Trp53 from let-7afd -/- and control Sftpc -tdT + cells after 2 months of iTAM (n = 4 samples per group from pools of 2 mice). Data are mean±s.e.m. *p < 0.05, by unpaired Student’s t test. f Quantification of AGER + Sftpc -tdT + in total Sftpc -tdT + cells from lungs of let-7afd AT2 and control mice at 1-or 5-months after iTAM. Data are mean±s.e.m, by unpaired Student’s t test. All panels are representative of three independent experiments.

    Journal: bioRxiv

    Article Title: Let-7 restrains an oncogenic epigenetic circuit in AT2 cells to prevent ectopic formation of fibrogenic transitional cell intermediates and pulmonary fibrosis

    doi: 10.1101/2024.05.22.595205

    Figure Lengend Snippet: a Immunostaining for γH2AX (green), SFTPC (red), DAPI (blue) upper panels; active CASP3 (green), SFTPC (red), AGER (gray), DAPI (blue) middle panels; AGER (green), Sftpc -tdT (red), DAPI (blue) lower panels in lungs of let-7afd AT2 and control mice after 1 month of iTAM. Arrows indicate γH2AX + SFTPC + cells (upper panel) or active-CASP3 + SFTPC + cells (middle panels). Scale bars: 25 μm. b - c Quantification of γH2AX + SFTPC + cells ( b ) or active-CASP3 + SFTPC + cells ( c ) in total SFTPC + cells (n = 3 mice per group). Data are mean±s.e.m. ****p < 0.0001, **p < 0.01, by unpaired Student’s t test. d Representative β-galactosidase staining in lungs of let-7afd AT2 and control mice after 1-month of iTAM. Arrowheads point to AT2 cells in thickened alveolar septa (n = 6 mice per group). Scale bars: 50 μm. e qPCR detection of Trp53 from let-7afd -/- and control Sftpc -tdT + cells after 2 months of iTAM (n = 4 samples per group from pools of 2 mice). Data are mean±s.e.m. *p < 0.05, by unpaired Student’s t test. f Quantification of AGER + Sftpc -tdT + in total Sftpc -tdT + cells from lungs of let-7afd AT2 and control mice at 1-or 5-months after iTAM. Data are mean±s.e.m, by unpaired Student’s t test. All panels are representative of three independent experiments.

    Article Snippet: Samples were then incubated overnight at 4°C with the following primary antibodies diluted in 1% Normal Goat Serum: Rat anti-Ki67 conjugated FITC (1:50, Invitrogen, 11-5698-82), Mouse anti-active/pro-caspase3 (1:20, Invitrogen, MA1-91637), Mouse anti-EZH2 (1:100, Invitrogen, 14-9867-82), Rabbit anti-BACH1 (1:200, Novus Biological, NBP2-55113), Mouse anti-γH2AX (1:200, Novus Biological, NB100-74435), Rabbit anti-CLDN4 (1:200, Invitrogen, 36-4800), Rabbit anti-Phospho-p70 S6K (1:200, Cell Signaling, 9234), Rabbit anti-pro-SFTPC (1:200, Millipore, AB3786), Rabbit anti-RFP/tdTomato (1:200, Rockland, 600-401-379), Rat anti-Krt8/TROMA-I (1:20, DSHB, TROMA-I-s), Rat anti-Galectin-3/LGALS3 (1:500, Cedarlane, CL8942AP), Rat anti-RAGE/AGER (1:100, R&D Systems, MAB1179-100), Rabbit anti-H3K27me (1:200, Invitrogen, MA5-11198), Rabbit anti-Aqp5 (1:100, Invitrogen, PA5-36529), Rabbit anti-Caveolin-1 (1:200, Cell Signaling, 3267), and Mouse anti-HopX (1:200, Santa Cruz, sc-398703).

    Techniques: Immunostaining, Staining

    Mitotic telomere deprotection is suppressed by WRN helicase independently of its catalytic activities. ( a ) Immunoblot of WRN in IMR-90 E6E7 hTERT cells transduced with shWRN or shScramble for 5 days. GAPDH serves as a loading control. ( b ) Representative images of meta-TIF assay from WRN knockdown cells after treatment with 100 ng/ml colcemid. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci per chromosome spread in indicated conditions. Violin plots illustrate the distribution of all data and averages from three independent experiments (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Kruskal–Wallis followed by Dunn’s test). ( d ) Immunoblot of WRN in IMR-90 E6E7 hTERT cells expressing exogenous WRN RES or Vector. Cells were transduced with shScramble or shWRN for 5 days before analysis. GAPDH serves as a loading control. ( e ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in c (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Kruskal–Wallis followed by Dunn’s test). ( f ) Immunoblot of WRN in cells expressing WRN RES and indicated WRN mutants. Transduced cells were harvested on day 10 post-infection. GAPDH serves as a loading control. ( g ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.

    Journal: Scientific Reports

    Article Title: A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection

    doi: 10.1038/s41598-023-27598-0

    Figure Lengend Snippet: Mitotic telomere deprotection is suppressed by WRN helicase independently of its catalytic activities. ( a ) Immunoblot of WRN in IMR-90 E6E7 hTERT cells transduced with shWRN or shScramble for 5 days. GAPDH serves as a loading control. ( b ) Representative images of meta-TIF assay from WRN knockdown cells after treatment with 100 ng/ml colcemid. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci per chromosome spread in indicated conditions. Violin plots illustrate the distribution of all data and averages from three independent experiments (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Kruskal–Wallis followed by Dunn’s test). ( d ) Immunoblot of WRN in IMR-90 E6E7 hTERT cells expressing exogenous WRN RES or Vector. Cells were transduced with shScramble or shWRN for 5 days before analysis. GAPDH serves as a loading control. ( e ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in c (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Kruskal–Wallis followed by Dunn’s test). ( f ) Immunoblot of WRN in cells expressing WRN RES and indicated WRN mutants. Transduced cells were harvested on day 10 post-infection. GAPDH serves as a loading control. ( g ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.

    Article Snippet: Samples were incubated with mouse anti-γ-H2AX p-Ser139 antibody (613,402 Clone 2F3, Biolegend) at 1:200 dilution in ABDIL buffer.

    Techniques: Western Blot, Transduction, Expressing, Plasmid Preparation, Infection

    WRN N-terminus coiled-coil region is sufficient to suppress MAD-TIFs. ( a ) Schematic representation of NLS and 4xFLAG tagged WRN fragments and derivative sub-fragments from the N-terminal WRN 2–499 fragment. Exo, exonuclease domain; Coiled, coiled-coil motif; Helicase, helicase domain; RQC, RecQ C-terminal DNA-binding domain; HRDC, helicase and RNaseD C-terminal domain. ( b ) Immunoblot of endogenous WRN and NLS-4FL-WRN fragments in IMR-90 E6E7 hTERT cells expressing indicated WRN fragments. Transduced cells were analyzed on day 10 post-infection. A black arrowhead indicates bands of the expected size of 60–70 kDa. GAPDH serves as a loading control. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). ( d ) Immunoblot of FLAG-WRN fragments in IMR-90 E6E7 hTERT cells expressing indicated WRN fragments. Transduced cells were harvested on day 12 post-infection. A black arrowhead indicates the expected fragment size (~ 27 kDa). Potential truncation, complex formation, and post-translational modifications are specified with white arrowheads. Asterisks represent unspecific bands from empty Vector. GAPDH serves as a loading control. ( e ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). ( f ) Immunoblot of FLAG-WRN fragments in IMR-90 E6E7 hTERT cells expressing indicated WRN fragments. Transduced cells were harvested on day 12 post-infection. Magenta and blue arrowheads indicate the expected size for the WRN168-333 (~ 27 kDa) and the WRN251-333 (~ 17 kDa) fragments, respectively. White arrowheads with colored lines indicate possible post-translational modifications or complex formation for each mutant. ( g ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.

    Journal: Scientific Reports

    Article Title: A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection

    doi: 10.1038/s41598-023-27598-0

    Figure Lengend Snippet: WRN N-terminus coiled-coil region is sufficient to suppress MAD-TIFs. ( a ) Schematic representation of NLS and 4xFLAG tagged WRN fragments and derivative sub-fragments from the N-terminal WRN 2–499 fragment. Exo, exonuclease domain; Coiled, coiled-coil motif; Helicase, helicase domain; RQC, RecQ C-terminal DNA-binding domain; HRDC, helicase and RNaseD C-terminal domain. ( b ) Immunoblot of endogenous WRN and NLS-4FL-WRN fragments in IMR-90 E6E7 hTERT cells expressing indicated WRN fragments. Transduced cells were analyzed on day 10 post-infection. A black arrowhead indicates bands of the expected size of 60–70 kDa. GAPDH serves as a loading control. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). ( d ) Immunoblot of FLAG-WRN fragments in IMR-90 E6E7 hTERT cells expressing indicated WRN fragments. Transduced cells were harvested on day 12 post-infection. A black arrowhead indicates the expected fragment size (~ 27 kDa). Potential truncation, complex formation, and post-translational modifications are specified with white arrowheads. Asterisks represent unspecific bands from empty Vector. GAPDH serves as a loading control. ( e ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). ( f ) Immunoblot of FLAG-WRN fragments in IMR-90 E6E7 hTERT cells expressing indicated WRN fragments. Transduced cells were harvested on day 12 post-infection. Magenta and blue arrowheads indicate the expected size for the WRN168-333 (~ 27 kDa) and the WRN251-333 (~ 17 kDa) fragments, respectively. White arrowheads with colored lines indicate possible post-translational modifications or complex formation for each mutant. ( g ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.

    Article Snippet: Samples were incubated with mouse anti-γ-H2AX p-Ser139 antibody (613,402 Clone 2F3, Biolegend) at 1:200 dilution in ABDIL buffer.

    Techniques: Binding Assay, Western Blot, Expressing, Infection, Plasmid Preparation, Mutagenesis

    WRN N-terminus does not perturb mitotic Aurora B or ATM kinase activities. ( a ) Distribution of mitotic duration in cells expressing indicated WRN fragments (Median, 25th, and 75th percentile; Kruskal–Wallis followed by Dunn's test). Cells were exposed to 500 nM taxol and analyzed by live-cell imaging data. Vector cells were co-treated with 40 nM Hesperadin as a control. ( b ) Ratio of each cell fate after mitosis in indicated cells. Results are separately shown as categorized by the duration of mitotic arrest. Mitosis longer than 2 h was defined as mitotic arrest. ( c ) Schematic of the timing of 100 ng/ml colcemid and 0.2 µg/ml bleomycin treatment in IMR-90 E6E7 hTERT cells expressing NLS-4xFL-WRN 2–499 fragment. ( d ) Representative images of γ-H2AX foci on mitotic chromosomes in cells treated as in ( c ). Images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( e ) Quantification of total γ-H2AX foci on mitotic chromosomes. Violin plots illustrate the distribution of all data and averages from three independent experiments (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test).

    Journal: Scientific Reports

    Article Title: A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection

    doi: 10.1038/s41598-023-27598-0

    Figure Lengend Snippet: WRN N-terminus does not perturb mitotic Aurora B or ATM kinase activities. ( a ) Distribution of mitotic duration in cells expressing indicated WRN fragments (Median, 25th, and 75th percentile; Kruskal–Wallis followed by Dunn's test). Cells were exposed to 500 nM taxol and analyzed by live-cell imaging data. Vector cells were co-treated with 40 nM Hesperadin as a control. ( b ) Ratio of each cell fate after mitosis in indicated cells. Results are separately shown as categorized by the duration of mitotic arrest. Mitosis longer than 2 h was defined as mitotic arrest. ( c ) Schematic of the timing of 100 ng/ml colcemid and 0.2 µg/ml bleomycin treatment in IMR-90 E6E7 hTERT cells expressing NLS-4xFL-WRN 2–499 fragment. ( d ) Representative images of γ-H2AX foci on mitotic chromosomes in cells treated as in ( c ). Images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( e ) Quantification of total γ-H2AX foci on mitotic chromosomes. Violin plots illustrate the distribution of all data and averages from three independent experiments (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test).

    Article Snippet: Samples were incubated with mouse anti-γ-H2AX p-Ser139 antibody (613,402 Clone 2F3, Biolegend) at 1:200 dilution in ABDIL buffer.

    Techniques: Expressing, Live Cell Imaging, Plasmid Preparation

    WRN supports TRF2 function to protect mitotic telomeres. ( a ) Immunoblot of endogenous WRN, FLAG-WRN fragments, and TRF2 upon TRF2 depletion in indicated cells. IMR-90 E6E7 hTERT cells expressing indicated WRN fragments were transduced with shTRF2 lentivirus and analyzed on day 7 post-infection. Blue-colored arrowhead indicates the expected size for WRN 168–333 (~ 27 kDa). White arrowheads (magenta line border for WRN 2–499 fragment and blue border for WRN 168–333 ) indicate possible post-translational modifications or complex formation. GAPDH serves as a loading control. ( b ) Representative images of meta-TIF assay from TRF2 knockdown cells expressing indicated WRN fragments after treatment with 100 ng/ml colcemid. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Mann–Whitney test). Dashed lines discriminate between the average number of TIFs generated in the interphase due to shTRF2 (2 h colcemid) and MAD-TIFs caused by mitotic arrest (24 h colcemid). ( d ) Immunoblot of endogenous WRN and TRF2 in indicated cells. IMR-90 E6E7 hTERT cells expressing exogenous TRF2 were transduced with shWRN lentivirus and analyzed on day 5 post-infection. GAPDH serves as a loading control. ( e ) Representative images of meta-TIF assay in indicated cells from d after treatment with 100 ng/ml colcemid for 24 h. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( f ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.

    Journal: Scientific Reports

    Article Title: A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection

    doi: 10.1038/s41598-023-27598-0

    Figure Lengend Snippet: WRN supports TRF2 function to protect mitotic telomeres. ( a ) Immunoblot of endogenous WRN, FLAG-WRN fragments, and TRF2 upon TRF2 depletion in indicated cells. IMR-90 E6E7 hTERT cells expressing indicated WRN fragments were transduced with shTRF2 lentivirus and analyzed on day 7 post-infection. Blue-colored arrowhead indicates the expected size for WRN 168–333 (~ 27 kDa). White arrowheads (magenta line border for WRN 2–499 fragment and blue border for WRN 168–333 ) indicate possible post-translational modifications or complex formation. GAPDH serves as a loading control. ( b ) Representative images of meta-TIF assay from TRF2 knockdown cells expressing indicated WRN fragments after treatment with 100 ng/ml colcemid. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( c ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 15/experiment for 2 h colcemid; n = 30/experiment for 24 h colcemid; mean ± s.e.m.; Mann–Whitney test). Dashed lines discriminate between the average number of TIFs generated in the interphase due to shTRF2 (2 h colcemid) and MAD-TIFs caused by mitotic arrest (24 h colcemid). ( d ) Immunoblot of endogenous WRN and TRF2 in indicated cells. IMR-90 E6E7 hTERT cells expressing exogenous TRF2 were transduced with shWRN lentivirus and analyzed on day 5 post-infection. GAPDH serves as a loading control. ( e ) Representative images of meta-TIF assay in indicated cells from d after treatment with 100 ng/ml colcemid for 24 h. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( f ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.

    Article Snippet: Samples were incubated with mouse anti-γ-H2AX p-Ser139 antibody (613,402 Clone 2F3, Biolegend) at 1:200 dilution in ABDIL buffer.

    Techniques: Western Blot, Expressing, Transduction, Infection, MANN-WHITNEY, Generated

    Phosphomimetic mutation at S282 of WRN 168–333 disrupts its MAD-TIF suppressor effect. ( a ) Schematic representation of four potential Aurora B sites in the 168–333 aa of WRN N-terminus. Sites of alanine and phosphomimetic mutations are indicated below. All mutants contain N-terminal NLS and 4xFLAG tags. ( b ) Immunoblot of FLAG-WRN fragments in indicated cells. Transduced cells were harvested on day 12 post-infection. A black arrowhead indicates the expected band size for all the mutants (~ 27 kDa), and white arrowheads indicate additional bands. ( c ) Representative images of meta-TIF assay in indicated cells after treatment with 100 ng/ml colcemid for 24 h. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( d ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.

    Journal: Scientific Reports

    Article Title: A non-catalytic N-terminus domain of WRN prevents mitotic telomere deprotection

    doi: 10.1038/s41598-023-27598-0

    Figure Lengend Snippet: Phosphomimetic mutation at S282 of WRN 168–333 disrupts its MAD-TIF suppressor effect. ( a ) Schematic representation of four potential Aurora B sites in the 168–333 aa of WRN N-terminus. Sites of alanine and phosphomimetic mutations are indicated below. All mutants contain N-terminal NLS and 4xFLAG tags. ( b ) Immunoblot of FLAG-WRN fragments in indicated cells. Transduced cells were harvested on day 12 post-infection. A black arrowhead indicates the expected band size for all the mutants (~ 27 kDa), and white arrowheads indicate additional bands. ( c ) Representative images of meta-TIF assay in indicated cells after treatment with 100 ng/ml colcemid for 24 h. The images show DAPI (blue), γ-H2AX (red), and telomere FISH (green). Scale bar, 10 µm. ( d ) Quantification of telomeric signals colocalized with γ-H2AX foci in indicated conditions as shown in Fig. c (n = 30/experiment; mean ± s.e.m.; Kruskal–Wallis followed by Dunn's test). Unprocessed blot images are shown in Supplementary Information files.

    Article Snippet: Samples were incubated with mouse anti-γ-H2AX p-Ser139 antibody (613,402 Clone 2F3, Biolegend) at 1:200 dilution in ABDIL buffer.

    Techniques: Mutagenesis, Western Blot, Infection

    Down-regulation of CK2 results in persistent γ-H2AX signal and reduced colony formation in human M059K glioblastoma cells . A . Cells were transfected with scramble siRNA (si-Scr), CK2α or -α'-siRNA for 72 hours. Where indicated, 0.5 μg/ml neocarzinostatin (NCS) was added to the medium in the last 24 hours of incubation. Fixed cells were subsequently labeled with anti-γ-H2AX antibody and with a FITC-conjugated secondary antibody. Nuclei were visualized by DAPI staining. B . Quantification of γ-H2AX-positive cells was performed by using ImageJ software and expressed as percentage of the total number of cells in each sample. Bars indicate mean values +/- standard deviation (SD) from three independent experiments. *P < 0.0001 indicates statistically significant difference in the number of γ-H2AX-positive cells in CK2-depleted versus si-Scr-treated cells. C . Cells were transfected with CK2α-, -α'-siRNA or scramble siRNA for 72 hours. 0.5 μg/ml NCS was added in the last hour of incubation. Control, refers to cell treated with transfection reagent only. Cells were allowed to form clusters for 14 days. Colonies were visualized by staining with crystal violet as described in Experimental Procedures. Bar graph shows cell colonies quantification. Average values +/- SD from three independent experiments are shown relative to control (i.e. transfection reagent-treated) cells. *P < 0.005 denotes statistically significant difference in number of colonies formed as compared to si-Scr-tranfected and NCS treated cells.

    Journal: BMC Molecular Biology

    Article Title: Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

    doi: 10.1186/1471-2199-13-7

    Figure Lengend Snippet: Down-regulation of CK2 results in persistent γ-H2AX signal and reduced colony formation in human M059K glioblastoma cells . A . Cells were transfected with scramble siRNA (si-Scr), CK2α or -α'-siRNA for 72 hours. Where indicated, 0.5 μg/ml neocarzinostatin (NCS) was added to the medium in the last 24 hours of incubation. Fixed cells were subsequently labeled with anti-γ-H2AX antibody and with a FITC-conjugated secondary antibody. Nuclei were visualized by DAPI staining. B . Quantification of γ-H2AX-positive cells was performed by using ImageJ software and expressed as percentage of the total number of cells in each sample. Bars indicate mean values +/- standard deviation (SD) from three independent experiments. *P < 0.0001 indicates statistically significant difference in the number of γ-H2AX-positive cells in CK2-depleted versus si-Scr-treated cells. C . Cells were transfected with CK2α-, -α'-siRNA or scramble siRNA for 72 hours. 0.5 μg/ml NCS was added in the last hour of incubation. Control, refers to cell treated with transfection reagent only. Cells were allowed to form clusters for 14 days. Colonies were visualized by staining with crystal violet as described in Experimental Procedures. Bar graph shows cell colonies quantification. Average values +/- SD from three independent experiments are shown relative to control (i.e. transfection reagent-treated) cells. *P < 0.005 denotes statistically significant difference in number of colonies formed as compared to si-Scr-tranfected and NCS treated cells.

    Article Snippet: For the analysis of γ-H2AX/CK2α' interaction by in situ PLA, cells grown on cover slips were incubated with mouse monoclonal anti-γ-H2AX (p-Ser139) antibody (#11174, Abcam) and a rabbit polyclonal antibody against CK2α'.

    Techniques: Transfection, Incubation, Labeling, Staining, Software, Standard Deviation

    CK2 co-localizes with γ-H2AX to sites of DNA damage . A . Association between CK2α' and γ-H2AX was revealed by in situ PLA. Cells expressing or lacking CK2α' were left untreated or incubated with 0.5 μg/ml NCS for 1 hour before fixation and labeling with primary antibodies directed against the indicated proteins. Control, refers to cells transfected with si-CK2α' and stained with secondary antibodies. Cell nuclei were revealed by Hoechst staining. B . Quantification of the number of signals/cell as distinct fluorescent red spots was performed by computer-assisted image analysis as described in Experimental Procedures. Mean values +/- SD from three independent experiments are shown. *P < 0.0001 denotes statistically significant difference.

    Journal: BMC Molecular Biology

    Article Title: Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

    doi: 10.1186/1471-2199-13-7

    Figure Lengend Snippet: CK2 co-localizes with γ-H2AX to sites of DNA damage . A . Association between CK2α' and γ-H2AX was revealed by in situ PLA. Cells expressing or lacking CK2α' were left untreated or incubated with 0.5 μg/ml NCS for 1 hour before fixation and labeling with primary antibodies directed against the indicated proteins. Control, refers to cells transfected with si-CK2α' and stained with secondary antibodies. Cell nuclei were revealed by Hoechst staining. B . Quantification of the number of signals/cell as distinct fluorescent red spots was performed by computer-assisted image analysis as described in Experimental Procedures. Mean values +/- SD from three independent experiments are shown. *P < 0.0001 denotes statistically significant difference.

    Article Snippet: For the analysis of γ-H2AX/CK2α' interaction by in situ PLA, cells grown on cover slips were incubated with mouse monoclonal anti-γ-H2AX (p-Ser139) antibody (#11174, Abcam) and a rabbit polyclonal antibody against CK2α'.

    Techniques: In Situ, Expressing, Incubation, Labeling, Transfection, Staining

    Cells overexpressing CK2 exhibit a prompt response to induction of DNA double-strand break . A . M059K cells transiently overexpressing CK2α'-DsRed or DsRed-fluorescent protein were incubated with 0.5 μg/ml NCS for the indicated times. Subsequently, cells were fixed and labeled with anti-γ-H2AX antibody. Cell nuclei were revealed by DAPI staining. Arrows: cells overexpressing CK2α'-DsRed are more efficient in DNA damage repair as compared to cells that do not overexpress the kinase. One representative experiment is shown. B . Average γ-H2AX foci number per red fluorescent cell as described in A . Mean values +/- SD from three independent experiments are shown. More than 100 cells have been analyzed per experiment. *P < 0.001 denotes statistically significant difference. NS, not significant.

    Journal: BMC Molecular Biology

    Article Title: Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

    doi: 10.1186/1471-2199-13-7

    Figure Lengend Snippet: Cells overexpressing CK2 exhibit a prompt response to induction of DNA double-strand break . A . M059K cells transiently overexpressing CK2α'-DsRed or DsRed-fluorescent protein were incubated with 0.5 μg/ml NCS for the indicated times. Subsequently, cells were fixed and labeled with anti-γ-H2AX antibody. Cell nuclei were revealed by DAPI staining. Arrows: cells overexpressing CK2α'-DsRed are more efficient in DNA damage repair as compared to cells that do not overexpress the kinase. One representative experiment is shown. B . Average γ-H2AX foci number per red fluorescent cell as described in A . Mean values +/- SD from three independent experiments are shown. More than 100 cells have been analyzed per experiment. *P < 0.001 denotes statistically significant difference. NS, not significant.

    Article Snippet: For the analysis of γ-H2AX/CK2α' interaction by in situ PLA, cells grown on cover slips were incubated with mouse monoclonal anti-γ-H2AX (p-Ser139) antibody (#11174, Abcam) and a rabbit polyclonal antibody against CK2α'.

    Techniques: Incubation, Labeling, Staining

    S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of p-H2ax in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

    Journal: Antioxidants

    Article Title: Sphingosine-1-Phosphate Alleviates Irradiation Induced Salivary Gland Hypofunction through Preserving Endothelial Cells and Resident Macrophages

    doi: 10.3390/antiox11102050

    Figure Lengend Snippet: S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of p-H2ax in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

    Article Snippet: PVDF membranes were incubated overnight with primary antibodies including Aqp5 (0.1 μg/mL, ab78486, Abcam, Waltham, MA, USA), Bax (1:1000, Abcam ab32503), Bcl2 (1:2000, Abcam ab182858), p-H2ax (1:1000, CST 80312, Danvers, MA, USA), cleaved Caspase3 (1:1000, CST 9664P), p53 (1:1000, 10442-1-AP, Proteintech, Rosemont, IL, USA), Nox4 (1:1000, Proteintech 14347), Nrf2 (1:1000, Proteintech 16396), Akt (1:1000, Proteintech 10176-2), p-Akt (1:2000, CST 4060T), Sod2 (1:1000, Abcam ab137037) eNOS (1:500, Abcam ab76198), p-eNOS (1:1000, CST 9570), and β-actin (1:50,000, Abclone AC026).

    Techniques: Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

    Effects of S1P on endothelial cells and macrophages in radiated salivary glands. ( A – D ) Expression of S1pr1 and Cd31 in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( E , F ) Levels of p-H2ax in Cd31 + endothelial cells in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( G – I ) Expression of F4/80 and levels of p-H2ax in F4/80 + macrophages in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( J ) mRNA levels of macrophage-enriched Hgf and endothelia-enriched Kitl in SMGs collected at 7 days after IR were examined with RT-qPCR. Quantified data are shown as mean ± SD, N = 3, NS: not significant vs. NT, ND: not detected, # : p < 0.05 vs. NT, *: p < 0.05, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

    Journal: Antioxidants

    Article Title: Sphingosine-1-Phosphate Alleviates Irradiation Induced Salivary Gland Hypofunction through Preserving Endothelial Cells and Resident Macrophages

    doi: 10.3390/antiox11102050

    Figure Lengend Snippet: Effects of S1P on endothelial cells and macrophages in radiated salivary glands. ( A – D ) Expression of S1pr1 and Cd31 in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( E , F ) Levels of p-H2ax in Cd31 + endothelial cells in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( G – I ) Expression of F4/80 and levels of p-H2ax in F4/80 + macrophages in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( J ) mRNA levels of macrophage-enriched Hgf and endothelia-enriched Kitl in SMGs collected at 7 days after IR were examined with RT-qPCR. Quantified data are shown as mean ± SD, N = 3, NS: not significant vs. NT, ND: not detected, # : p < 0.05 vs. NT, *: p < 0.05, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

    Article Snippet: PVDF membranes were incubated overnight with primary antibodies including Aqp5 (0.1 μg/mL, ab78486, Abcam, Waltham, MA, USA), Bax (1:1000, Abcam ab32503), Bcl2 (1:2000, Abcam ab182858), p-H2ax (1:1000, CST 80312, Danvers, MA, USA), cleaved Caspase3 (1:1000, CST 9664P), p53 (1:1000, 10442-1-AP, Proteintech, Rosemont, IL, USA), Nox4 (1:1000, Proteintech 14347), Nrf2 (1:1000, Proteintech 16396), Akt (1:1000, Proteintech 10176-2), p-Akt (1:2000, CST 4060T), Sod2 (1:1000, Abcam ab137037) eNOS (1:500, Abcam ab76198), p-eNOS (1:1000, CST 9570), and β-actin (1:50,000, Abclone AC026).

    Techniques: Expressing, Double Immunofluorescence Staining, Quantitative RT-PCR

    S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of p-H2ax in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

    Journal: Antioxidants

    Article Title: Sphingosine-1-Phosphate Alleviates Irradiation Induced Salivary Gland Hypofunction through Preserving Endothelial Cells and Resident Macrophages

    doi: 10.3390/antiox11102050

    Figure Lengend Snippet: S1P decreased oxidative stress and DNA damage in radiated salivary glands. ( A – D ) Levels of p-H2ax in SMGs collected at 4 or 24 h after IR were examined with immunofluorescence staining and Western blot analyses. Magnification: ×400. ( E – H ) Levels of Sod2, Nox4 and Nrf2 proteins in SMGs were examined with Western blot. ( I ) Sod activities in SMG homogenate were examined with a commercial kit. ( J ) mRNA levels of Nrf2 target genes in SMGs collected at 24 h after IR were examined with RT-qPCR. Data are shown as mean ± SD, N = 3, ns: not significant vs. NT, #: p < 0.05 vs. NT, *: p < 0.05, **: p < 0.01, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

    Article Snippet: These samples were incubated overnight at 4 °C with following primary antibodies: anti-p-H2ax (1:200, 80312, CST, Danvers, MA, USA), anti-Nrf2 (1:50, 16396, Proteintech, Rosemont, IL, USA), anti-S1pr1 (1:500, PA11040, ThermoFisher Scientific, Waltham, MA, USA), anti-Cd31 (1:50, ab281583, Abcam, Waltham, MA, USA), and anti-F4/80 (1:100, bs7058R, Bioss, Beijing, China).

    Techniques: Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

    Effects of S1P on endothelial cells and macrophages in radiated salivary glands. ( A – D ) Expression of S1pr1 and Cd31 in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( E , F ) Levels of p-H2ax in Cd31 + endothelial cells in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( G – I ) Expression of F4/80 and levels of p-H2ax in F4/80 + macrophages in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( J ) mRNA levels of macrophage-enriched Hgf and endothelia-enriched Kitl in SMGs collected at 7 days after IR were examined with RT-qPCR. Quantified data are shown as mean ± SD, N = 3, NS: not significant vs. NT, ND: not detected, # : p < 0.05 vs. NT, *: p < 0.05, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

    Journal: Antioxidants

    Article Title: Sphingosine-1-Phosphate Alleviates Irradiation Induced Salivary Gland Hypofunction through Preserving Endothelial Cells and Resident Macrophages

    doi: 10.3390/antiox11102050

    Figure Lengend Snippet: Effects of S1P on endothelial cells and macrophages in radiated salivary glands. ( A – D ) Expression of S1pr1 and Cd31 in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( E , F ) Levels of p-H2ax in Cd31 + endothelial cells in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( G – I ) Expression of F4/80 and levels of p-H2ax in F4/80 + macrophages in SMGs collected at 7 days after IR were examined with double immunofluorescence staining and quantified. ( J ) mRNA levels of macrophage-enriched Hgf and endothelia-enriched Kitl in SMGs collected at 7 days after IR were examined with RT-qPCR. Quantified data are shown as mean ± SD, N = 3, NS: not significant vs. NT, ND: not detected, # : p < 0.05 vs. NT, *: p < 0.05, ***: p < 0.001. Quantitative data were analyzed using one-way ANOVA with Tukey’s multiple comparisons.

    Article Snippet: These samples were incubated overnight at 4 °C with following primary antibodies: anti-p-H2ax (1:200, 80312, CST, Danvers, MA, USA), anti-Nrf2 (1:50, 16396, Proteintech, Rosemont, IL, USA), anti-S1pr1 (1:500, PA11040, ThermoFisher Scientific, Waltham, MA, USA), anti-Cd31 (1:50, ab281583, Abcam, Waltham, MA, USA), and anti-F4/80 (1:100, bs7058R, Bioss, Beijing, China).

    Techniques: Expressing, Double Immunofluorescence Staining, Quantitative RT-PCR