rabbit anti chk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti chk1
    The ability of NVP-AUY922 to abrogate G 2 arrest in the presence of dsDNA damage was investigated by western blot and FACS analysis in all four cell lines (p53 status: HCT116 p53wt, Hela HPV-positive, HN5 and HN3 p53 mutant). (A) HeLa and HN3 cells were pre-treated with NVP-AUY922 for 16 h at the concentrations indicated. Cells were then mock irradiated or irradiated with 4 Gy and 4 h later whole cell lysates harvested, 30 µg protein resolved on 10% SDS-PAGE then probed for phospho-H2ax, phospho-histone H3 and <t>phospho-CHK1</t> by western blot. (B) The effect of NVP-AUY922 on depletion of total-CHK1 was also investigated in unirradiated cells with γ-tubulin probed as loading control. (C+D) Cells were exposed to the NVP-AUY922 concentrations indicated for 16 h before mock irradiation or irradiation with 4 Gy. Cells were fixed 9 h and 48 h post-irradiation before staining for the mitotic marker p-histone H3 and DNA content with propidium iodide. SubG 1 and >4N populations were quantified by FACS analysis. Data represents ± SEM of three independent experiments each recording at least 10,000 events. Statistical analysis carried out by two-tailed t-test between the groups indicated, *p<0.05 **p<0.01 ***p<0.001.
    Rabbit Anti Chk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The HSP90 Inhibitor NVP-AUY922 Radiosensitizes by Abrogation of Homologous Recombination Resulting in Mitotic Entry with Unresolved DNA Damage"

    Article Title: The HSP90 Inhibitor NVP-AUY922 Radiosensitizes by Abrogation of Homologous Recombination Resulting in Mitotic Entry with Unresolved DNA Damage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035436

    The ability of NVP-AUY922 to abrogate G 2 arrest in the presence of dsDNA damage was investigated by western blot and FACS analysis in all four cell lines (p53 status: HCT116 p53wt, Hela HPV-positive, HN5 and HN3 p53 mutant). (A) HeLa and HN3 cells were pre-treated with NVP-AUY922 for 16 h at the concentrations indicated. Cells were then mock irradiated or irradiated with 4 Gy and 4 h later whole cell lysates harvested, 30 µg protein resolved on 10% SDS-PAGE then probed for phospho-H2ax, phospho-histone H3 and phospho-CHK1 by western blot. (B) The effect of NVP-AUY922 on depletion of total-CHK1 was also investigated in unirradiated cells with γ-tubulin probed as loading control. (C+D) Cells were exposed to the NVP-AUY922 concentrations indicated for 16 h before mock irradiation or irradiation with 4 Gy. Cells were fixed 9 h and 48 h post-irradiation before staining for the mitotic marker p-histone H3 and DNA content with propidium iodide. SubG 1 and >4N populations were quantified by FACS analysis. Data represents ± SEM of three independent experiments each recording at least 10,000 events. Statistical analysis carried out by two-tailed t-test between the groups indicated, *p<0.05 **p<0.01 ***p<0.001.
    Figure Legend Snippet: The ability of NVP-AUY922 to abrogate G 2 arrest in the presence of dsDNA damage was investigated by western blot and FACS analysis in all four cell lines (p53 status: HCT116 p53wt, Hela HPV-positive, HN5 and HN3 p53 mutant). (A) HeLa and HN3 cells were pre-treated with NVP-AUY922 for 16 h at the concentrations indicated. Cells were then mock irradiated or irradiated with 4 Gy and 4 h later whole cell lysates harvested, 30 µg protein resolved on 10% SDS-PAGE then probed for phospho-H2ax, phospho-histone H3 and phospho-CHK1 by western blot. (B) The effect of NVP-AUY922 on depletion of total-CHK1 was also investigated in unirradiated cells with γ-tubulin probed as loading control. (C+D) Cells were exposed to the NVP-AUY922 concentrations indicated for 16 h before mock irradiation or irradiation with 4 Gy. Cells were fixed 9 h and 48 h post-irradiation before staining for the mitotic marker p-histone H3 and DNA content with propidium iodide. SubG 1 and >4N populations were quantified by FACS analysis. Data represents ± SEM of three independent experiments each recording at least 10,000 events. Statistical analysis carried out by two-tailed t-test between the groups indicated, *p<0.05 **p<0.01 ***p<0.001.

    Techniques Used: Western Blot, Mutagenesis, Irradiation, SDS Page, Staining, Marker, Two Tailed Test

    rabbit anti chk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti chk1
    The ability of NVP-AUY922 to abrogate G 2 arrest in the presence of dsDNA damage was investigated by western blot and FACS analysis in all four cell lines (p53 status: HCT116 p53wt, Hela HPV-positive, HN5 and HN3 p53 mutant). (A) HeLa and HN3 cells were pre-treated with NVP-AUY922 for 16 h at the concentrations indicated. Cells were then mock irradiated or irradiated with 4 Gy and 4 h later whole cell lysates harvested, 30 µg protein resolved on 10% SDS-PAGE then probed for phospho-H2ax, phospho-histone H3 and <t>phospho-CHK1</t> by western blot. (B) The effect of NVP-AUY922 on depletion of total-CHK1 was also investigated in unirradiated cells with γ-tubulin probed as loading control. (C+D) Cells were exposed to the NVP-AUY922 concentrations indicated for 16 h before mock irradiation or irradiation with 4 Gy. Cells were fixed 9 h and 48 h post-irradiation before staining for the mitotic marker p-histone H3 and DNA content with propidium iodide. SubG 1 and >4N populations were quantified by FACS analysis. Data represents ± SEM of three independent experiments each recording at least 10,000 events. Statistical analysis carried out by two-tailed t-test between the groups indicated, *p<0.05 **p<0.01 ***p<0.001.
    Rabbit Anti Chk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The HSP90 Inhibitor NVP-AUY922 Radiosensitizes by Abrogation of Homologous Recombination Resulting in Mitotic Entry with Unresolved DNA Damage"

    Article Title: The HSP90 Inhibitor NVP-AUY922 Radiosensitizes by Abrogation of Homologous Recombination Resulting in Mitotic Entry with Unresolved DNA Damage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035436

    The ability of NVP-AUY922 to abrogate G 2 arrest in the presence of dsDNA damage was investigated by western blot and FACS analysis in all four cell lines (p53 status: HCT116 p53wt, Hela HPV-positive, HN5 and HN3 p53 mutant). (A) HeLa and HN3 cells were pre-treated with NVP-AUY922 for 16 h at the concentrations indicated. Cells were then mock irradiated or irradiated with 4 Gy and 4 h later whole cell lysates harvested, 30 µg protein resolved on 10% SDS-PAGE then probed for phospho-H2ax, phospho-histone H3 and phospho-CHK1 by western blot. (B) The effect of NVP-AUY922 on depletion of total-CHK1 was also investigated in unirradiated cells with γ-tubulin probed as loading control. (C+D) Cells were exposed to the NVP-AUY922 concentrations indicated for 16 h before mock irradiation or irradiation with 4 Gy. Cells were fixed 9 h and 48 h post-irradiation before staining for the mitotic marker p-histone H3 and DNA content with propidium iodide. SubG 1 and >4N populations were quantified by FACS analysis. Data represents ± SEM of three independent experiments each recording at least 10,000 events. Statistical analysis carried out by two-tailed t-test between the groups indicated, *p<0.05 **p<0.01 ***p<0.001.
    Figure Legend Snippet: The ability of NVP-AUY922 to abrogate G 2 arrest in the presence of dsDNA damage was investigated by western blot and FACS analysis in all four cell lines (p53 status: HCT116 p53wt, Hela HPV-positive, HN5 and HN3 p53 mutant). (A) HeLa and HN3 cells were pre-treated with NVP-AUY922 for 16 h at the concentrations indicated. Cells were then mock irradiated or irradiated with 4 Gy and 4 h later whole cell lysates harvested, 30 µg protein resolved on 10% SDS-PAGE then probed for phospho-H2ax, phospho-histone H3 and phospho-CHK1 by western blot. (B) The effect of NVP-AUY922 on depletion of total-CHK1 was also investigated in unirradiated cells with γ-tubulin probed as loading control. (C+D) Cells were exposed to the NVP-AUY922 concentrations indicated for 16 h before mock irradiation or irradiation with 4 Gy. Cells were fixed 9 h and 48 h post-irradiation before staining for the mitotic marker p-histone H3 and DNA content with propidium iodide. SubG 1 and >4N populations were quantified by FACS analysis. Data represents ± SEM of three independent experiments each recording at least 10,000 events. Statistical analysis carried out by two-tailed t-test between the groups indicated, *p<0.05 **p<0.01 ***p<0.001.

    Techniques Used: Western Blot, Mutagenesis, Irradiation, SDS Page, Staining, Marker, Two Tailed Test

    rabbit anti chk1  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc rabbit anti chk1
    Unless otherwise stated, the following results were repeated with three independent experiments. (A) Whole embryo extracts at E7.5 were prepared for Western blotting analysis. (B) RT-PCR for embryos prepared from E7.5. Note lower levels of α-tubulin, <t>Chk1,</t> and Chk2, but not β-tubulin, in mp29 GT/GT embryos. GAPDH was used as an input control. (C) Total RNAs from embryos at E7.5 were extracted and first strand cDNAs were transcribed for quantitative real-time PCR analysis to determine the relative pre-mRNA and mRNA levels of α-tubulin and Chk1. GAPDH was used as a normalized control. (D) NIH3T3 cells were co-transfected with siRNAs and E1A splicing reporter for in vivo alternative splicing assay. Total RNAs were isolated and subjected to RT-PCR. The splicing products were quantitatively analyzed with three independent experiments. GAPDH was used as a normalized control. * indicated p <0.05 in one-way ANOVA F-test.
    Rabbit Anti Chk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Disruption of Murine mp29/Syf2/Ntc31 Gene Results in Embryonic Lethality with Aberrant Checkpoint Response"

    Article Title: Disruption of Murine mp29/Syf2/Ntc31 Gene Results in Embryonic Lethality with Aberrant Checkpoint Response

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033538

    Unless otherwise stated, the following results were repeated with three independent experiments. (A) Whole embryo extracts at E7.5 were prepared for Western blotting analysis. (B) RT-PCR for embryos prepared from E7.5. Note lower levels of α-tubulin, Chk1, and Chk2, but not β-tubulin, in mp29 GT/GT embryos. GAPDH was used as an input control. (C) Total RNAs from embryos at E7.5 were extracted and first strand cDNAs were transcribed for quantitative real-time PCR analysis to determine the relative pre-mRNA and mRNA levels of α-tubulin and Chk1. GAPDH was used as a normalized control. (D) NIH3T3 cells were co-transfected with siRNAs and E1A splicing reporter for in vivo alternative splicing assay. Total RNAs were isolated and subjected to RT-PCR. The splicing products were quantitatively analyzed with three independent experiments. GAPDH was used as a normalized control. * indicated p <0.05 in one-way ANOVA F-test.
    Figure Legend Snippet: Unless otherwise stated, the following results were repeated with three independent experiments. (A) Whole embryo extracts at E7.5 were prepared for Western blotting analysis. (B) RT-PCR for embryos prepared from E7.5. Note lower levels of α-tubulin, Chk1, and Chk2, but not β-tubulin, in mp29 GT/GT embryos. GAPDH was used as an input control. (C) Total RNAs from embryos at E7.5 were extracted and first strand cDNAs were transcribed for quantitative real-time PCR analysis to determine the relative pre-mRNA and mRNA levels of α-tubulin and Chk1. GAPDH was used as a normalized control. (D) NIH3T3 cells were co-transfected with siRNAs and E1A splicing reporter for in vivo alternative splicing assay. Total RNAs were isolated and subjected to RT-PCR. The splicing products were quantitatively analyzed with three independent experiments. GAPDH was used as a normalized control. * indicated p <0.05 in one-way ANOVA F-test.

    Techniques Used: Western Blot, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Transfection, In Vivo, Splicing Assay, Isolation

    mp29 GT/+ mice were mated with mp29 Tg/+ mice to generate mp29 GT/+ mp29 Tg/+ littermates. Inbreed of mp29 GT/+ mp29 Tg/+ male mice with mp29 GT/+ mp29 +/+ female mice gave birth to mp29 GT/GT mp29 Tg/+ mice. (A) Genotyping of complemented mice by PCR analysis. NSPF1/NeoES PCR products indicated the presence of U3NeoSV1 vector. NSPF1/NSPR1 primers were used to determine whether both of alleles were inserted with U3NeoSV1 vector. mPGKF1/mp29R1 PCR products indicated the presence of mp29 transgene with a product of 500 bp. Tg refers to the hemizygous mp29 transgene. (B) The genotyping of 76 complemented offsprings was examined. Eight pups exhibited homozygously interrupted genotype but with mp29 transgene ( mp29 GT/GT mp29 Tg/+ ). There were no mp29 GT/GT mice that could survive without the presence of mp29 transgene. (C) Tissue extracts of the livers from complemented mice were prepared for Western blot analysis. Note that normal expression levels of α-tubulin, Chk1, and Chk2 were restored in mp29 GT/GT mp29 Tg/+ mice.
    Figure Legend Snippet: mp29 GT/+ mice were mated with mp29 Tg/+ mice to generate mp29 GT/+ mp29 Tg/+ littermates. Inbreed of mp29 GT/+ mp29 Tg/+ male mice with mp29 GT/+ mp29 +/+ female mice gave birth to mp29 GT/GT mp29 Tg/+ mice. (A) Genotyping of complemented mice by PCR analysis. NSPF1/NeoES PCR products indicated the presence of U3NeoSV1 vector. NSPF1/NSPR1 primers were used to determine whether both of alleles were inserted with U3NeoSV1 vector. mPGKF1/mp29R1 PCR products indicated the presence of mp29 transgene with a product of 500 bp. Tg refers to the hemizygous mp29 transgene. (B) The genotyping of 76 complemented offsprings was examined. Eight pups exhibited homozygously interrupted genotype but with mp29 transgene ( mp29 GT/GT mp29 Tg/+ ). There were no mp29 GT/GT mice that could survive without the presence of mp29 transgene. (C) Tissue extracts of the livers from complemented mice were prepared for Western blot analysis. Note that normal expression levels of α-tubulin, Chk1, and Chk2 were restored in mp29 GT/GT mp29 Tg/+ mice.

    Techniques Used: Plasmid Preparation, Western Blot, Expressing

    anti phospho chk1 wasfrom cell signaling technology  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho chk1 wasfrom cell signaling technology
    Unsynchronized HeLa cells were treated with 0, 20, or 30 µM DhL and lysed or fixed at the indicated times. (A) Immunoblot analysis of phospho-ATM (p-ATM) and <t>phospho-Chk1</t> <t>(p-Chk1).</t> Representative assay of 3 independent experiments. β-actin was employed as a loading control. Cells were stained with DAPI to visualize the nuclei and treated with specific antibodies for γH2AX (B) and 53BP1 (C). Left: representative fields from 24 h treatment. Insets: magnification of the areas indicated by boxes in the top row. Representative fields for 48 h treatment are shown in . Right: quantification of the number of cells with more than 10 γH2AX foci and more than 5 53BP1 foci. At least 200 nuclei were scored for each sample. Bar: 10 µm. Data represent mean ± SEM of 3 independent experiments. * p≤0.05, ** p≤0.01 vs. control group (0 µM DhL).
    Anti Phospho Chk1 Wasfrom Cell Signaling Technology, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The Sesquiterpene Lactone Dehydroleucodine Triggers Senescence and Apoptosis in Association with Accumulation of DNA Damage Markers"

    Article Title: The Sesquiterpene Lactone Dehydroleucodine Triggers Senescence and Apoptosis in Association with Accumulation of DNA Damage Markers

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053168

    Unsynchronized HeLa cells were treated with 0, 20, or 30 µM DhL and lysed or fixed at the indicated times. (A) Immunoblot analysis of phospho-ATM (p-ATM) and phospho-Chk1 (p-Chk1). Representative assay of 3 independent experiments. β-actin was employed as a loading control. Cells were stained with DAPI to visualize the nuclei and treated with specific antibodies for γH2AX (B) and 53BP1 (C). Left: representative fields from 24 h treatment. Insets: magnification of the areas indicated by boxes in the top row. Representative fields for 48 h treatment are shown in . Right: quantification of the number of cells with more than 10 γH2AX foci and more than 5 53BP1 foci. At least 200 nuclei were scored for each sample. Bar: 10 µm. Data represent mean ± SEM of 3 independent experiments. * p≤0.05, ** p≤0.01 vs. control group (0 µM DhL).
    Figure Legend Snippet: Unsynchronized HeLa cells were treated with 0, 20, or 30 µM DhL and lysed or fixed at the indicated times. (A) Immunoblot analysis of phospho-ATM (p-ATM) and phospho-Chk1 (p-Chk1). Representative assay of 3 independent experiments. β-actin was employed as a loading control. Cells were stained with DAPI to visualize the nuclei and treated with specific antibodies for γH2AX (B) and 53BP1 (C). Left: representative fields from 24 h treatment. Insets: magnification of the areas indicated by boxes in the top row. Representative fields for 48 h treatment are shown in . Right: quantification of the number of cells with more than 10 γH2AX foci and more than 5 53BP1 foci. At least 200 nuclei were scored for each sample. Bar: 10 µm. Data represent mean ± SEM of 3 independent experiments. * p≤0.05, ** p≤0.01 vs. control group (0 µM DhL).

    Techniques Used: Western Blot, Staining

    phosphorylated ser 317 chk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phosphorylated ser 317 chk1
    (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of <t>pCHK1/CHK1</t> and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.
    Phosphorylated Ser 317 Chk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Mutation of Serine 1333 in the ATR HEAT Repeats Creates a Hyperactive Kinase"

    Article Title: Mutation of Serine 1333 in the ATR HEAT Repeats Creates a Hyperactive Kinase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0099397

    (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of pCHK1/CHK1 and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.
    Figure Legend Snippet: (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of pCHK1/CHK1 and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.

    Techniques Used: Clone Assay, Expressing, SDS Page, Western Blot

    rabbit anti phospho chk1 ser317  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti phospho chk1 ser317
    Mcph1 -Δe8 cells exhibit PCC and defective DDR. ( A ) Representative images of PCC in Mcph1 -Δe8 MEFs. Primary MEF cells were stained with a pS10-H3 antibody (red) and counterstained with DAPI (blue). ( B ) Quantification of the percentages of PCC cells (prophase cells lacking the pS10-H3 signal) in control and Mcph1 -Δe8 primary MEF cells. More than 250 prophase cells were scored in each group of the indicated genotype. ( C ) Western blot analysis of <t>p-Chk1</t> and γH2AX in control and Mcph1 -Δe8 MEFs with or without HU treatment. β-actin was used as a loading control. ( D ) The quantification of the indicated protein intensities from panel ( C ). Unpaired Student’s t -test was used for statistical analysis. ***, p < 0.001.
    Rabbit Anti Phospho Chk1 Ser317, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Central Domain of MCPH1 Controls Development of the Cerebral Cortex and Gonads in Mice"

    Article Title: The Central Domain of MCPH1 Controls Development of the Cerebral Cortex and Gonads in Mice

    Journal: Cells

    doi: 10.3390/cells11172715

    Mcph1 -Δe8 cells exhibit PCC and defective DDR. ( A ) Representative images of PCC in Mcph1 -Δe8 MEFs. Primary MEF cells were stained with a pS10-H3 antibody (red) and counterstained with DAPI (blue). ( B ) Quantification of the percentages of PCC cells (prophase cells lacking the pS10-H3 signal) in control and Mcph1 -Δe8 primary MEF cells. More than 250 prophase cells were scored in each group of the indicated genotype. ( C ) Western blot analysis of p-Chk1 and γH2AX in control and Mcph1 -Δe8 MEFs with or without HU treatment. β-actin was used as a loading control. ( D ) The quantification of the indicated protein intensities from panel ( C ). Unpaired Student’s t -test was used for statistical analysis. ***, p < 0.001.
    Figure Legend Snippet: Mcph1 -Δe8 cells exhibit PCC and defective DDR. ( A ) Representative images of PCC in Mcph1 -Δe8 MEFs. Primary MEF cells were stained with a pS10-H3 antibody (red) and counterstained with DAPI (blue). ( B ) Quantification of the percentages of PCC cells (prophase cells lacking the pS10-H3 signal) in control and Mcph1 -Δe8 primary MEF cells. More than 250 prophase cells were scored in each group of the indicated genotype. ( C ) Western blot analysis of p-Chk1 and γH2AX in control and Mcph1 -Δe8 MEFs with or without HU treatment. β-actin was used as a loading control. ( D ) The quantification of the indicated protein intensities from panel ( C ). Unpaired Student’s t -test was used for statistical analysis. ***, p < 0.001.

    Techniques Used: Staining, Western Blot

    rabbit anti chk 1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti chk 1
    Irreparable damage is not channeled for apoptosis due to timing defects in ATM/ATR signaling. ( A ) Quantification of the number of pachytene nuclei with CED-1::GFP engulfment. Each data point reflects the number of nuclei in one gonad completely engulfed by CED-1::GFP. Black lines indicate median. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). ( B ) Quantification of the number of nuclei with acridine orange staining in the indicated backgrounds. Each data point reflects the number of acridine orange-positive nuclei in one gonad. Statistical significance calculated by Mann–Whitney t -test. ( C ) Quantification of the number of late pachytene nuclei with (+) or without (−) PGL-1 staining. Each data point reflects the number of nuclei within one gonad. Error bars reflect mean with SEM. Statistical comparisons drawn between quantifications of the same category (e.g. PGL-1(+) v. PGL-1(+)) by Mann–Whitney t-test. ( D ) Quantification of the number of FLAG::RPA-2 foci across the germline. X-axis denotes the zone of the germline and the y-axis reflects the average number of FLAG foci. Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). ( E ) Measurements of ATM/ATR signaling. Fluorescence intensity of (pS/pT) QG signal across the gonad. X-axis denotes the zone of the germline and the y-axis denotes arbitrary fluorescent units per μm 2 . Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). Gray asterisks reflect significance compared to wild type, purple asterisks to syp-3 , and pink asterisks to rnh-1.0; rnh-2 . ( F ) Quantification of the number of <t>CHK-1</t> foci in late pachytene nuclei. Error bars reflect mean with SEM. Statistical significance calculated by Mann–Whitney t -test. ( G ) Fluorescence intensity of CEP-1::GFP signal in late pachytene nuclei. Each data point reflects the fluorescence intensity of one LP nucleus with correction to the average background signal for that gonad. Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05).
    Rabbit Anti Chk 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "R-loop-induced irreparable DNA damage evades checkpoint detection in the C. elegans germline"

    Article Title: R-loop-induced irreparable DNA damage evades checkpoint detection in the C. elegans germline

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkac621

    Irreparable damage is not channeled for apoptosis due to timing defects in ATM/ATR signaling. ( A ) Quantification of the number of pachytene nuclei with CED-1::GFP engulfment. Each data point reflects the number of nuclei in one gonad completely engulfed by CED-1::GFP. Black lines indicate median. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). ( B ) Quantification of the number of nuclei with acridine orange staining in the indicated backgrounds. Each data point reflects the number of acridine orange-positive nuclei in one gonad. Statistical significance calculated by Mann–Whitney t -test. ( C ) Quantification of the number of late pachytene nuclei with (+) or without (−) PGL-1 staining. Each data point reflects the number of nuclei within one gonad. Error bars reflect mean with SEM. Statistical comparisons drawn between quantifications of the same category (e.g. PGL-1(+) v. PGL-1(+)) by Mann–Whitney t-test. ( D ) Quantification of the number of FLAG::RPA-2 foci across the germline. X-axis denotes the zone of the germline and the y-axis reflects the average number of FLAG foci. Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). ( E ) Measurements of ATM/ATR signaling. Fluorescence intensity of (pS/pT) QG signal across the gonad. X-axis denotes the zone of the germline and the y-axis denotes arbitrary fluorescent units per μm 2 . Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). Gray asterisks reflect significance compared to wild type, purple asterisks to syp-3 , and pink asterisks to rnh-1.0; rnh-2 . ( F ) Quantification of the number of CHK-1 foci in late pachytene nuclei. Error bars reflect mean with SEM. Statistical significance calculated by Mann–Whitney t -test. ( G ) Fluorescence intensity of CEP-1::GFP signal in late pachytene nuclei. Each data point reflects the fluorescence intensity of one LP nucleus with correction to the average background signal for that gonad. Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05).
    Figure Legend Snippet: Irreparable damage is not channeled for apoptosis due to timing defects in ATM/ATR signaling. ( A ) Quantification of the number of pachytene nuclei with CED-1::GFP engulfment. Each data point reflects the number of nuclei in one gonad completely engulfed by CED-1::GFP. Black lines indicate median. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). ( B ) Quantification of the number of nuclei with acridine orange staining in the indicated backgrounds. Each data point reflects the number of acridine orange-positive nuclei in one gonad. Statistical significance calculated by Mann–Whitney t -test. ( C ) Quantification of the number of late pachytene nuclei with (+) or without (−) PGL-1 staining. Each data point reflects the number of nuclei within one gonad. Error bars reflect mean with SEM. Statistical comparisons drawn between quantifications of the same category (e.g. PGL-1(+) v. PGL-1(+)) by Mann–Whitney t-test. ( D ) Quantification of the number of FLAG::RPA-2 foci across the germline. X-axis denotes the zone of the germline and the y-axis reflects the average number of FLAG foci. Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). ( E ) Measurements of ATM/ATR signaling. Fluorescence intensity of (pS/pT) QG signal across the gonad. X-axis denotes the zone of the germline and the y-axis denotes arbitrary fluorescent units per μm 2 . Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). Gray asterisks reflect significance compared to wild type, purple asterisks to syp-3 , and pink asterisks to rnh-1.0; rnh-2 . ( F ) Quantification of the number of CHK-1 foci in late pachytene nuclei. Error bars reflect mean with SEM. Statistical significance calculated by Mann–Whitney t -test. ( G ) Fluorescence intensity of CEP-1::GFP signal in late pachytene nuclei. Each data point reflects the fluorescence intensity of one LP nucleus with correction to the average background signal for that gonad. Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05).

    Techniques Used: MANN-WHITNEY, Staining, Fluorescence

    phosphorylated chk1 pchk1 at ser317 at ser317 s317  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phosphorylated chk1 pchk1 at ser317 at ser317 s317
    The control rad9A +/+ (+/+) and rad9A -/- (KO) PC3 cells transfected with vector (V), FLAG-WT-hRad9A (WT), FLAG-DM-hRad9A (DM), FLAG-LD-hRad9A (LD) or FLAG-K220A-hRad9A (K220A) were treated with 16 mM hydroxyurea (HU) for 2 hours and recovered for 2 hours or left untreated. Cell extracts were subjected to Western blotting analysis with antibodies against <t>phosphorylated</t> <t>Chk1</t> <t>(pChk1)</t> at <t>Ser317</t> (S-317) ( A ), total Chk1 ( B ), and β-actin ( C ). Molecular weight markers were loaded on lane 1 with indicated sizes in KDa.
    Phosphorylated Chk1 Pchk1 At Ser317 At Ser317 S317, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "DNA binding by the Rad9A subunit of the Rad9-Rad1-Hus1 complex"

    Article Title: DNA binding by the Rad9A subunit of the Rad9-Rad1-Hus1 complex

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0272645

    The control rad9A +/+ (+/+) and rad9A -/- (KO) PC3 cells transfected with vector (V), FLAG-WT-hRad9A (WT), FLAG-DM-hRad9A (DM), FLAG-LD-hRad9A (LD) or FLAG-K220A-hRad9A (K220A) were treated with 16 mM hydroxyurea (HU) for 2 hours and recovered for 2 hours or left untreated. Cell extracts were subjected to Western blotting analysis with antibodies against phosphorylated Chk1 (pChk1) at Ser317 (S-317) ( A ), total Chk1 ( B ), and β-actin ( C ). Molecular weight markers were loaded on lane 1 with indicated sizes in KDa.
    Figure Legend Snippet: The control rad9A +/+ (+/+) and rad9A -/- (KO) PC3 cells transfected with vector (V), FLAG-WT-hRad9A (WT), FLAG-DM-hRad9A (DM), FLAG-LD-hRad9A (LD) or FLAG-K220A-hRad9A (K220A) were treated with 16 mM hydroxyurea (HU) for 2 hours and recovered for 2 hours or left untreated. Cell extracts were subjected to Western blotting analysis with antibodies against phosphorylated Chk1 (pChk1) at Ser317 (S-317) ( A ), total Chk1 ( B ), and β-actin ( C ). Molecular weight markers were loaded on lane 1 with indicated sizes in KDa.

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Molecular Weight

    pchk1 s317  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pchk1 s317
    Pchk1 S317, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti chk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti chk1
    RNF4 depletion was associated with a cell cycle defect. (A) RNF4-depleted cells exhibited increases in fraction of cells in the G1 phase and decreases in fraction in S phase. Cell cycle distributions were determined by incorporation of BrdU for 20 min, anti-BrdU and propidium iodide staining, followed by flow cytometric analysis. Results of three independent experiments were averaged and standard deviations shown. (B) RNF4-depleted U2OS cells exhibited similar levels of <t>CHK1</t> phosphorylation (Ser317) with or without treatment with 2 mM HU for 16 h. Levels were also measured in cells blocked in HU for 16 h then released into normal medium for various times. (C) RNF4-depleted HCT116 cells exhibited similar cell cycle distributions compared to control-depleted cells with or without treatment with HU, caffeine, or both. Results of three independent experiments were averaged and standard deviations shown.
    Rabbit Anti Chk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "RNF4 Regulates the BLM Helicase in Recovery From Replication Fork Collapse"

    Article Title: RNF4 Regulates the BLM Helicase in Recovery From Replication Fork Collapse

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2021.753535

    RNF4 depletion was associated with a cell cycle defect. (A) RNF4-depleted cells exhibited increases in fraction of cells in the G1 phase and decreases in fraction in S phase. Cell cycle distributions were determined by incorporation of BrdU for 20 min, anti-BrdU and propidium iodide staining, followed by flow cytometric analysis. Results of three independent experiments were averaged and standard deviations shown. (B) RNF4-depleted U2OS cells exhibited similar levels of CHK1 phosphorylation (Ser317) with or without treatment with 2 mM HU for 16 h. Levels were also measured in cells blocked in HU for 16 h then released into normal medium for various times. (C) RNF4-depleted HCT116 cells exhibited similar cell cycle distributions compared to control-depleted cells with or without treatment with HU, caffeine, or both. Results of three independent experiments were averaged and standard deviations shown.
    Figure Legend Snippet: RNF4 depletion was associated with a cell cycle defect. (A) RNF4-depleted cells exhibited increases in fraction of cells in the G1 phase and decreases in fraction in S phase. Cell cycle distributions were determined by incorporation of BrdU for 20 min, anti-BrdU and propidium iodide staining, followed by flow cytometric analysis. Results of three independent experiments were averaged and standard deviations shown. (B) RNF4-depleted U2OS cells exhibited similar levels of CHK1 phosphorylation (Ser317) with or without treatment with 2 mM HU for 16 h. Levels were also measured in cells blocked in HU for 16 h then released into normal medium for various times. (C) RNF4-depleted HCT116 cells exhibited similar cell cycle distributions compared to control-depleted cells with or without treatment with HU, caffeine, or both. Results of three independent experiments were averaged and standard deviations shown.

    Techniques Used: Staining

    rabbit anti phospho chk1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti phospho chk1
    ( A ) A diagram of the third instar larva showing the dorsal trunk (DT) of the second thoracic metamere (Tr2, colored in green and marked by dashed line). The diagram also shows the timecourse of G2 arrest and cell division in Tr2. The cells in Tr2 DT remain geographically isolated from tracheal cells in other branches during larval life. ( B–E ) Levels of the ROS reporter 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCFDA) in Tr2 DT during larval stages. Shown in the figures are H 2 DCFDA staining in L2 ( B ), 0–8 hr L3 ( C ), 16–24 hr L3 ( D ), and 32–40 hr L3 ( E ) in wild type ( btl-Gal4 ) animals. ( F–I ) Levels of the ROS reporter dihydroethidium (DHE) in Tr2 DT during larval stages. Shown in the figures are DHE staining in L2 ( F ), 0–8 hr L3 ( G ), 16–24 hr L3 ( H ), and 32–40 hr L3 ( I ) in wild type ( btl-Gal4 ) animals. ( J, K ) Effect of btl -Gal4-dependent overexpression of superoxide dismutase 1 (SOD1) on levels of ROS reporters in Tr2 DT. ( J ) H 2 DCFDA staining in btl-SOD1 ( btl-GAL4/UAS-SOD1 )-expressing larvae (n ≥ 6 tracheae per condition per timepoint). ( K ) DHE staining in btl-SOD1 larvae (n ≥ 6 tracheae per condition per timepoint). ( L ) Effect of SOD1 overexpression on cell numbers in Tr2 DT at different larval stages. Graph shows numbers of Tr2 tracheoblasts in wild type ( btl-Gal4), btl-SOD1 (btl-GAL4/UAS-SOD1 ), and <t>btl-Chk1</t> RNAi (btl-GAL4/UAS-Chk1 RNAi ) larvae at L2, 0–8 hr L3, 16–24 hr L3, 32–40 hr L3, and wandering L3 (WL3) (n ≥ 7 tracheae per condition per timepoint). ( M ) Effect of SOD1 overexpression on mitotic indices in Tr2 DT (see text). Graph shows mitotic indices in Tr2 DT in wild type and btl-SOD1 (btl-GAL4/UAS-SOD1 )-expressing larvae at L2, 0–8 hr L3 and 16–24 hr L3 (mean values ± standard deviation, n ≥ 7 tracheae per condition per timepoint). ( N ) Effect of SOD1 overexpression on Chk1 phosphorylation in Tr2 tracheoblasts. Shown in the figure is phosphorylated Chk1 (pChk1, phospho-Chk1Ser 345 ) immunostaining (red) in Tr2 DT in wild type ( btl-GAL4 ) and btl-SOD1 (btl-GAL4/UAS-SOD1 ) larvae at L2. Scale bars = 10 µm. Dashed lines outline the cuticular lumen of the tracheal tube here and elsewhere and are shifted outward to include the epithelial lining when they overlap with the signal. Student’s t-test: *p<0.00001. Figure 1—source data 1. Cell Frequencies in SOD1 overexpressing animals. Figure 1—source data 2. Mitotic indices in SOD1 overexpressing animals.
    Rabbit Anti Phospho Chk1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts"

    Article Title: Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts

    Journal: eLife

    doi: 10.7554/eLife.68636

    ( A ) A diagram of the third instar larva showing the dorsal trunk (DT) of the second thoracic metamere (Tr2, colored in green and marked by dashed line). The diagram also shows the timecourse of G2 arrest and cell division in Tr2. The cells in Tr2 DT remain geographically isolated from tracheal cells in other branches during larval life. ( B–E ) Levels of the ROS reporter 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCFDA) in Tr2 DT during larval stages. Shown in the figures are H 2 DCFDA staining in L2 ( B ), 0–8 hr L3 ( C ), 16–24 hr L3 ( D ), and 32–40 hr L3 ( E ) in wild type ( btl-Gal4 ) animals. ( F–I ) Levels of the ROS reporter dihydroethidium (DHE) in Tr2 DT during larval stages. Shown in the figures are DHE staining in L2 ( F ), 0–8 hr L3 ( G ), 16–24 hr L3 ( H ), and 32–40 hr L3 ( I ) in wild type ( btl-Gal4 ) animals. ( J, K ) Effect of btl -Gal4-dependent overexpression of superoxide dismutase 1 (SOD1) on levels of ROS reporters in Tr2 DT. ( J ) H 2 DCFDA staining in btl-SOD1 ( btl-GAL4/UAS-SOD1 )-expressing larvae (n ≥ 6 tracheae per condition per timepoint). ( K ) DHE staining in btl-SOD1 larvae (n ≥ 6 tracheae per condition per timepoint). ( L ) Effect of SOD1 overexpression on cell numbers in Tr2 DT at different larval stages. Graph shows numbers of Tr2 tracheoblasts in wild type ( btl-Gal4), btl-SOD1 (btl-GAL4/UAS-SOD1 ), and btl-Chk1 RNAi (btl-GAL4/UAS-Chk1 RNAi ) larvae at L2, 0–8 hr L3, 16–24 hr L3, 32–40 hr L3, and wandering L3 (WL3) (n ≥ 7 tracheae per condition per timepoint). ( M ) Effect of SOD1 overexpression on mitotic indices in Tr2 DT (see text). Graph shows mitotic indices in Tr2 DT in wild type and btl-SOD1 (btl-GAL4/UAS-SOD1 )-expressing larvae at L2, 0–8 hr L3 and 16–24 hr L3 (mean values ± standard deviation, n ≥ 7 tracheae per condition per timepoint). ( N ) Effect of SOD1 overexpression on Chk1 phosphorylation in Tr2 tracheoblasts. Shown in the figure is phosphorylated Chk1 (pChk1, phospho-Chk1Ser 345 ) immunostaining (red) in Tr2 DT in wild type ( btl-GAL4 ) and btl-SOD1 (btl-GAL4/UAS-SOD1 ) larvae at L2. Scale bars = 10 µm. Dashed lines outline the cuticular lumen of the tracheal tube here and elsewhere and are shifted outward to include the epithelial lining when they overlap with the signal. Student’s t-test: *p<0.00001. Figure 1—source data 1. Cell Frequencies in SOD1 overexpressing animals. Figure 1—source data 2. Mitotic indices in SOD1 overexpressing animals.
    Figure Legend Snippet: ( A ) A diagram of the third instar larva showing the dorsal trunk (DT) of the second thoracic metamere (Tr2, colored in green and marked by dashed line). The diagram also shows the timecourse of G2 arrest and cell division in Tr2. The cells in Tr2 DT remain geographically isolated from tracheal cells in other branches during larval life. ( B–E ) Levels of the ROS reporter 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCFDA) in Tr2 DT during larval stages. Shown in the figures are H 2 DCFDA staining in L2 ( B ), 0–8 hr L3 ( C ), 16–24 hr L3 ( D ), and 32–40 hr L3 ( E ) in wild type ( btl-Gal4 ) animals. ( F–I ) Levels of the ROS reporter dihydroethidium (DHE) in Tr2 DT during larval stages. Shown in the figures are DHE staining in L2 ( F ), 0–8 hr L3 ( G ), 16–24 hr L3 ( H ), and 32–40 hr L3 ( I ) in wild type ( btl-Gal4 ) animals. ( J, K ) Effect of btl -Gal4-dependent overexpression of superoxide dismutase 1 (SOD1) on levels of ROS reporters in Tr2 DT. ( J ) H 2 DCFDA staining in btl-SOD1 ( btl-GAL4/UAS-SOD1 )-expressing larvae (n ≥ 6 tracheae per condition per timepoint). ( K ) DHE staining in btl-SOD1 larvae (n ≥ 6 tracheae per condition per timepoint). ( L ) Effect of SOD1 overexpression on cell numbers in Tr2 DT at different larval stages. Graph shows numbers of Tr2 tracheoblasts in wild type ( btl-Gal4), btl-SOD1 (btl-GAL4/UAS-SOD1 ), and btl-Chk1 RNAi (btl-GAL4/UAS-Chk1 RNAi ) larvae at L2, 0–8 hr L3, 16–24 hr L3, 32–40 hr L3, and wandering L3 (WL3) (n ≥ 7 tracheae per condition per timepoint). ( M ) Effect of SOD1 overexpression on mitotic indices in Tr2 DT (see text). Graph shows mitotic indices in Tr2 DT in wild type and btl-SOD1 (btl-GAL4/UAS-SOD1 )-expressing larvae at L2, 0–8 hr L3 and 16–24 hr L3 (mean values ± standard deviation, n ≥ 7 tracheae per condition per timepoint). ( N ) Effect of SOD1 overexpression on Chk1 phosphorylation in Tr2 tracheoblasts. Shown in the figure is phosphorylated Chk1 (pChk1, phospho-Chk1Ser 345 ) immunostaining (red) in Tr2 DT in wild type ( btl-GAL4 ) and btl-SOD1 (btl-GAL4/UAS-SOD1 ) larvae at L2. Scale bars = 10 µm. Dashed lines outline the cuticular lumen of the tracheal tube here and elsewhere and are shifted outward to include the epithelial lining when they overlap with the signal. Student’s t-test: *p<0.00001. Figure 1—source data 1. Cell Frequencies in SOD1 overexpressing animals. Figure 1—source data 2. Mitotic indices in SOD1 overexpressing animals.

    Techniques Used: Isolation, Staining, Over Expression, Expressing, Standard Deviation, Immunostaining

    ( A–D ) Detailed analysis of DNA damage in Tr2 dorsal trunk (DT). Shown here are findings from three different reporters of genotoxic stress. ( A ) 8-Oxo-2'-deoxyguanosine (8-Oxo-dG) immunostaining in wild type ( btl-GAL4 ) Tr2 DT in untreated tracheae (left panel) and tracheae exposed to 1 mM H 2 O 2 for 30 min ex vivo (right panel) at L2. ( B ) 8-Oxo-dG immunostaining in wild type ( btl-GAL4 ) endocycling cells of the tracheae exposed to 1 mM H 2 O 2 for 30 min ex vivo at L2. ( C ) GFP immunostaining in non-irradiated and γ-irradiated larvae expressing RPA70-GFP. Shown in the figure are GFP immunostaining in non-irradiated larvae (top panel) and larvae exposed to 50 Gy of γ-radiation (bottom panel) at L2. ( D ) γ-H2AX Ser139 immunostaining in Tr2 DT in wild type ( btl-GAL4 ) non-irradiated larvae (top panel) and larvae irradiated with 50 Gy of γ-radiation (bottom panel) at L2. ( E–H ) Analysis of the contribution of components of the DNA damage-dependent activation of ATR/Chk1 to Chk1 activation in Tr2 DT. Effects of the knockdown of ATR, ATRIP, TOPBP1, and Claspin on phosphorylated checkpoint kinase 1 (pChk1) levels in Tr2 DT at L2. pChk1 immunostaining (red) in Tr2 DT in ( E ) btl-ATR RNAi ( btl-GAL4/UAS-ATR RNAi ), ( F ) btl-ATRIP RNAi ( btl-GAL4/UAS-ATRIP RNAi ), ( G ) btl-TOPBP1 RNAi ( btl-GAL4/+; UAS-TOPBP1 RNAi /+ ), and ( H ) btl-Claspin RNAi ( btl-GAL4/+; UAS-Claspin RNAi /+ ) larvae at L2. ( I ) Effects of knockdown of ATR, ATRIP, TOPBP1, and Claspin on cell numbers in Tr2 DT. Graph shows numbers of Tr2 tracheoblasts in wild type ( btl-Gal4 ), btl-ATR RNAi ( btl-GAL4/UAS-ATR RNAi ), btl-ATRIP RNAi ( btl-GAL4/UAS-ATRIP RNAi ), btl-TOPBP1 RNAi ( btl-GAL4/+; UAS-TOPBP1 RNAi /+ ), and btl-Claspin RNAi ( btl-GAL4/+; UAS-Claspin RNAi /+ ) at L2 and 16–24 hr L3 (mean values ± standard deviation, n ≥ 7 tracheae per condition per timepoint). Scale bars = 5 µm ( A–D ), 10 µm ( E–H ). Student’s t-test: *p<0.00001. Figure 4—source data 1. Cell frequencies in ATR RNAi , ATRIP RNAi , TOPBP1 RNAi and Claspin RNAi expressing animals.
    Figure Legend Snippet: ( A–D ) Detailed analysis of DNA damage in Tr2 dorsal trunk (DT). Shown here are findings from three different reporters of genotoxic stress. ( A ) 8-Oxo-2'-deoxyguanosine (8-Oxo-dG) immunostaining in wild type ( btl-GAL4 ) Tr2 DT in untreated tracheae (left panel) and tracheae exposed to 1 mM H 2 O 2 for 30 min ex vivo (right panel) at L2. ( B ) 8-Oxo-dG immunostaining in wild type ( btl-GAL4 ) endocycling cells of the tracheae exposed to 1 mM H 2 O 2 for 30 min ex vivo at L2. ( C ) GFP immunostaining in non-irradiated and γ-irradiated larvae expressing RPA70-GFP. Shown in the figure are GFP immunostaining in non-irradiated larvae (top panel) and larvae exposed to 50 Gy of γ-radiation (bottom panel) at L2. ( D ) γ-H2AX Ser139 immunostaining in Tr2 DT in wild type ( btl-GAL4 ) non-irradiated larvae (top panel) and larvae irradiated with 50 Gy of γ-radiation (bottom panel) at L2. ( E–H ) Analysis of the contribution of components of the DNA damage-dependent activation of ATR/Chk1 to Chk1 activation in Tr2 DT. Effects of the knockdown of ATR, ATRIP, TOPBP1, and Claspin on phosphorylated checkpoint kinase 1 (pChk1) levels in Tr2 DT at L2. pChk1 immunostaining (red) in Tr2 DT in ( E ) btl-ATR RNAi ( btl-GAL4/UAS-ATR RNAi ), ( F ) btl-ATRIP RNAi ( btl-GAL4/UAS-ATRIP RNAi ), ( G ) btl-TOPBP1 RNAi ( btl-GAL4/+; UAS-TOPBP1 RNAi /+ ), and ( H ) btl-Claspin RNAi ( btl-GAL4/+; UAS-Claspin RNAi /+ ) larvae at L2. ( I ) Effects of knockdown of ATR, ATRIP, TOPBP1, and Claspin on cell numbers in Tr2 DT. Graph shows numbers of Tr2 tracheoblasts in wild type ( btl-Gal4 ), btl-ATR RNAi ( btl-GAL4/UAS-ATR RNAi ), btl-ATRIP RNAi ( btl-GAL4/UAS-ATRIP RNAi ), btl-TOPBP1 RNAi ( btl-GAL4/+; UAS-TOPBP1 RNAi /+ ), and btl-Claspin RNAi ( btl-GAL4/+; UAS-Claspin RNAi /+ ) at L2 and 16–24 hr L3 (mean values ± standard deviation, n ≥ 7 tracheae per condition per timepoint). Scale bars = 5 µm ( A–D ), 10 µm ( E–H ). Student’s t-test: *p<0.00001. Figure 4—source data 1. Cell frequencies in ATR RNAi , ATRIP RNAi , TOPBP1 RNAi and Claspin RNAi expressing animals.

    Techniques Used: Immunostaining, Ex Vivo, Irradiation, Expressing, Activation Assay, Standard Deviation

    Effect of knockdown of Duox and ATRIP or TOPBP1 or Claspin on phosphorylated checkpoint kinase 1 (pChk1) levels in Tr2 dorsal trunk (DT) in larvae exposed to 50 Gy of γ-radiation at L2. ( A ) Schematic describing the protocol for exposure to γ-radiation and immunostaining. ( B–D ) pChk1 immunostaining in Tr2 DT in ( B ) btl-Duox RNAi , ATRIP RNAi (btl-GAL4/UAS-ATRIP RNAi ; UAS-Duox RNAi (32903)/+ ), ( C ) btl-Duox RNAi , TOPBP1 RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-TOPBP1 RNAi ), and ( D ) btl-Duox RNAi , Claspin RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-Claspin RNAi ) larvae at 1 hr post exposure to 50 Gy of γ-radiation at L2 (n ≥ 6 tracheae per condition). Scale bars = 10 µm.
    Figure Legend Snippet: Effect of knockdown of Duox and ATRIP or TOPBP1 or Claspin on phosphorylated checkpoint kinase 1 (pChk1) levels in Tr2 dorsal trunk (DT) in larvae exposed to 50 Gy of γ-radiation at L2. ( A ) Schematic describing the protocol for exposure to γ-radiation and immunostaining. ( B–D ) pChk1 immunostaining in Tr2 DT in ( B ) btl-Duox RNAi , ATRIP RNAi (btl-GAL4/UAS-ATRIP RNAi ; UAS-Duox RNAi (32903)/+ ), ( C ) btl-Duox RNAi , TOPBP1 RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-TOPBP1 RNAi ), and ( D ) btl-Duox RNAi , Claspin RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-Claspin RNAi ) larvae at 1 hr post exposure to 50 Gy of γ-radiation at L2 (n ≥ 6 tracheae per condition). Scale bars = 10 µm.

    Techniques Used: Immunostaining

    ( A–E ) Kinetics of Chk1 phosphorylation on exposure to γ-radiation. ( A ) Regimen for γ-irradiation and analysis of pChk1. pChk1 immunostaining (red) in Tr2 dorsal trunk (DT) in btl-Duox RNAi (btl-GAL4/+; UAS-Duox RNAi (32903/+ )) in non-irradiated larvae ( B ) and larvae exposed to with 50 Gy of γ-radiation after ( C ) 1 hr, ( D ) 30 min, and ( E ) 2 min post irradiation at L2. ( F ) Effect of knockdown of ATR on Chk1 activation in Tr2 DT in larvae exposed to γ-radiation. pChk1 immunostaining (red) in Tr2 DT in btl-ATR RNAi (btl-GAL4/UAS-ATR RNAi ) larvae exposed to 50 Gy of γ-radiation at 1 hr post exposure at L2 (n ≥ 6 tracheae per condition) Scale bars = 10 µm.
    Figure Legend Snippet: ( A–E ) Kinetics of Chk1 phosphorylation on exposure to γ-radiation. ( A ) Regimen for γ-irradiation and analysis of pChk1. pChk1 immunostaining (red) in Tr2 dorsal trunk (DT) in btl-Duox RNAi (btl-GAL4/+; UAS-Duox RNAi (32903/+ )) in non-irradiated larvae ( B ) and larvae exposed to with 50 Gy of γ-radiation after ( C ) 1 hr, ( D ) 30 min, and ( E ) 2 min post irradiation at L2. ( F ) Effect of knockdown of ATR on Chk1 activation in Tr2 DT in larvae exposed to γ-radiation. pChk1 immunostaining (red) in Tr2 DT in btl-ATR RNAi (btl-GAL4/UAS-ATR RNAi ) larvae exposed to 50 Gy of γ-radiation at 1 hr post exposure at L2 (n ≥ 6 tracheae per condition) Scale bars = 10 µm.

    Techniques Used: Irradiation, Immunostaining, Activation Assay

    ( A–E ) Kinetics of Chk1 phosphorylation upon exposure to H 2 O 2 ex vivo. ( A ) Regimen for H 2 O 2 treatment and analysis of pChk1 in Tr2 dorsal trunk (DT) in L2. pChk1 immunostaining (red) in Tr2 DT in ( B ) untreated btl-Duox RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/+ )-expressing tracheae and treated with 100 µM H 2 O 2 for ( C ) 30 min, ( D ) 5 min, and ( E ) 2 min. ( F ) Effect of knockdown of ATR on Chk1 activation in Tr2 DT upon exposure to H 2 O 2 ex vivo. pChk1 immunostaining (red) in Tr2 DT in btl-ATR RNAi (btl-GAL4/UAS-ATR RNAi ) tracheae treated with 100 µM H 2 O 2 for 30 min. ( G–I ) Effect of knockdown of Duox and ATRIP or TOPBP1 or Claspin on pChk1 levels in Tr2 DT in tracheae exposed to 100 µM H 2 O 2 at L2. pChk1 immunostaining (red) in Tr2 DT in ( G ) btl-Duox RNAi , ATRIP RNAi ( btl-GAL4/ UAS-ATRIP RNAi ; UAS-Duox RNAi (32903)/+ ), ( H ) btl -Duox RNAi , TOPBP1 RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-TOPBP1 RNAi ) and ( I ) btl-Duox RNAi , Claspin RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-Claspin RNAi ) tracheae treated with 100 µM H 2 O 2 for 2 min at L2. ( J ) Model for the regulation of ATR/Chk1 activation in Tr2 DT. We propose that H 2 O 2 can induce ATR-dependent phosphorylation and activation of Chk1 in the absence of detectable DNA damage, leading to G2 arrest in Tr2 tracheoblasts. Scale bars = 10 µm.
    Figure Legend Snippet: ( A–E ) Kinetics of Chk1 phosphorylation upon exposure to H 2 O 2 ex vivo. ( A ) Regimen for H 2 O 2 treatment and analysis of pChk1 in Tr2 dorsal trunk (DT) in L2. pChk1 immunostaining (red) in Tr2 DT in ( B ) untreated btl-Duox RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/+ )-expressing tracheae and treated with 100 µM H 2 O 2 for ( C ) 30 min, ( D ) 5 min, and ( E ) 2 min. ( F ) Effect of knockdown of ATR on Chk1 activation in Tr2 DT upon exposure to H 2 O 2 ex vivo. pChk1 immunostaining (red) in Tr2 DT in btl-ATR RNAi (btl-GAL4/UAS-ATR RNAi ) tracheae treated with 100 µM H 2 O 2 for 30 min. ( G–I ) Effect of knockdown of Duox and ATRIP or TOPBP1 or Claspin on pChk1 levels in Tr2 DT in tracheae exposed to 100 µM H 2 O 2 at L2. pChk1 immunostaining (red) in Tr2 DT in ( G ) btl-Duox RNAi , ATRIP RNAi ( btl-GAL4/ UAS-ATRIP RNAi ; UAS-Duox RNAi (32903)/+ ), ( H ) btl -Duox RNAi , TOPBP1 RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-TOPBP1 RNAi ) and ( I ) btl-Duox RNAi , Claspin RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-Claspin RNAi ) tracheae treated with 100 µM H 2 O 2 for 2 min at L2. ( J ) Model for the regulation of ATR/Chk1 activation in Tr2 DT. We propose that H 2 O 2 can induce ATR-dependent phosphorylation and activation of Chk1 in the absence of detectable DNA damage, leading to G2 arrest in Tr2 tracheoblasts. Scale bars = 10 µm.

    Techniques Used: Ex Vivo, Immunostaining, Expressing, Activation Assay


    Figure Legend Snippet:

    Techniques Used: Amplification


    Figure Legend Snippet:

    Techniques Used:


    Figure Legend Snippet:

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    Cell Signaling Technology Inc rabbit anti chk1
    The ability of NVP-AUY922 to abrogate G 2 arrest in the presence of dsDNA damage was investigated by western blot and FACS analysis in all four cell lines (p53 status: HCT116 p53wt, Hela HPV-positive, HN5 and HN3 p53 mutant). (A) HeLa and HN3 cells were pre-treated with NVP-AUY922 for 16 h at the concentrations indicated. Cells were then mock irradiated or irradiated with 4 Gy and 4 h later whole cell lysates harvested, 30 µg protein resolved on 10% SDS-PAGE then probed for phospho-H2ax, phospho-histone H3 and <t>phospho-CHK1</t> by western blot. (B) The effect of NVP-AUY922 on depletion of total-CHK1 was also investigated in unirradiated cells with γ-tubulin probed as loading control. (C+D) Cells were exposed to the NVP-AUY922 concentrations indicated for 16 h before mock irradiation or irradiation with 4 Gy. Cells were fixed 9 h and 48 h post-irradiation before staining for the mitotic marker p-histone H3 and DNA content with propidium iodide. SubG 1 and >4N populations were quantified by FACS analysis. Data represents ± SEM of three independent experiments each recording at least 10,000 events. Statistical analysis carried out by two-tailed t-test between the groups indicated, *p<0.05 **p<0.01 ***p<0.001.
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    Unsynchronized HeLa cells were treated with 0, 20, or 30 µM DhL and lysed or fixed at the indicated times. (A) Immunoblot analysis of phospho-ATM (p-ATM) and <t>phospho-Chk1</t> <t>(p-Chk1).</t> Representative assay of 3 independent experiments. β-actin was employed as a loading control. Cells were stained with DAPI to visualize the nuclei and treated with specific antibodies for γH2AX (B) and 53BP1 (C). Left: representative fields from 24 h treatment. Insets: magnification of the areas indicated by boxes in the top row. Representative fields for 48 h treatment are shown in . Right: quantification of the number of cells with more than 10 γH2AX foci and more than 5 53BP1 foci. At least 200 nuclei were scored for each sample. Bar: 10 µm. Data represent mean ± SEM of 3 independent experiments. * p≤0.05, ** p≤0.01 vs. control group (0 µM DhL).
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    Cell Signaling Technology Inc phosphorylated ser 317 chk1
    (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of <t>pCHK1/CHK1</t> and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.
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    Cell Signaling Technology Inc rabbit anti phospho chk1 ser317
    Mcph1 -Δe8 cells exhibit PCC and defective DDR. ( A ) Representative images of PCC in Mcph1 -Δe8 MEFs. Primary MEF cells were stained with a pS10-H3 antibody (red) and counterstained with DAPI (blue). ( B ) Quantification of the percentages of PCC cells (prophase cells lacking the pS10-H3 signal) in control and Mcph1 -Δe8 primary MEF cells. More than 250 prophase cells were scored in each group of the indicated genotype. ( C ) Western blot analysis of <t>p-Chk1</t> and γH2AX in control and Mcph1 -Δe8 MEFs with or without HU treatment. β-actin was used as a loading control. ( D ) The quantification of the indicated protein intensities from panel ( C ). Unpaired Student’s t -test was used for statistical analysis. ***, p < 0.001.
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    Cell Signaling Technology Inc rabbit anti chk 1
    Irreparable damage is not channeled for apoptosis due to timing defects in ATM/ATR signaling. ( A ) Quantification of the number of pachytene nuclei with CED-1::GFP engulfment. Each data point reflects the number of nuclei in one gonad completely engulfed by CED-1::GFP. Black lines indicate median. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). ( B ) Quantification of the number of nuclei with acridine orange staining in the indicated backgrounds. Each data point reflects the number of acridine orange-positive nuclei in one gonad. Statistical significance calculated by Mann–Whitney t -test. ( C ) Quantification of the number of late pachytene nuclei with (+) or without (−) PGL-1 staining. Each data point reflects the number of nuclei within one gonad. Error bars reflect mean with SEM. Statistical comparisons drawn between quantifications of the same category (e.g. PGL-1(+) v. PGL-1(+)) by Mann–Whitney t-test. ( D ) Quantification of the number of FLAG::RPA-2 foci across the germline. X-axis denotes the zone of the germline and the y-axis reflects the average number of FLAG foci. Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). ( E ) Measurements of ATM/ATR signaling. Fluorescence intensity of (pS/pT) QG signal across the gonad. X-axis denotes the zone of the germline and the y-axis denotes arbitrary fluorescent units per μm 2 . Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). Gray asterisks reflect significance compared to wild type, purple asterisks to syp-3 , and pink asterisks to rnh-1.0; rnh-2 . ( F ) Quantification of the number of <t>CHK-1</t> foci in late pachytene nuclei. Error bars reflect mean with SEM. Statistical significance calculated by Mann–Whitney t -test. ( G ) Fluorescence intensity of CEP-1::GFP signal in late pachytene nuclei. Each data point reflects the fluorescence intensity of one LP nucleus with correction to the average background signal for that gonad. Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05).
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    Cell Signaling Technology Inc phosphorylated chk1 pchk1 at ser317 at ser317 s317
    The control rad9A +/+ (+/+) and rad9A -/- (KO) PC3 cells transfected with vector (V), FLAG-WT-hRad9A (WT), FLAG-DM-hRad9A (DM), FLAG-LD-hRad9A (LD) or FLAG-K220A-hRad9A (K220A) were treated with 16 mM hydroxyurea (HU) for 2 hours and recovered for 2 hours or left untreated. Cell extracts were subjected to Western blotting analysis with antibodies against <t>phosphorylated</t> <t>Chk1</t> <t>(pChk1)</t> at <t>Ser317</t> (S-317) ( A ), total Chk1 ( B ), and β-actin ( C ). Molecular weight markers were loaded on lane 1 with indicated sizes in KDa.
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    The control rad9A +/+ (+/+) and rad9A -/- (KO) PC3 cells transfected with vector (V), FLAG-WT-hRad9A (WT), FLAG-DM-hRad9A (DM), FLAG-LD-hRad9A (LD) or FLAG-K220A-hRad9A (K220A) were treated with 16 mM hydroxyurea (HU) for 2 hours and recovered for 2 hours or left untreated. Cell extracts were subjected to Western blotting analysis with antibodies against <t>phosphorylated</t> <t>Chk1</t> <t>(pChk1)</t> at <t>Ser317</t> (S-317) ( A ), total Chk1 ( B ), and β-actin ( C ). Molecular weight markers were loaded on lane 1 with indicated sizes in KDa.
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    Cell Signaling Technology Inc rabbit anti phospho chk1
    ( A ) A diagram of the third instar larva showing the dorsal trunk (DT) of the second thoracic metamere (Tr2, colored in green and marked by dashed line). The diagram also shows the timecourse of G2 arrest and cell division in Tr2. The cells in Tr2 DT remain geographically isolated from tracheal cells in other branches during larval life. ( B–E ) Levels of the ROS reporter 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCFDA) in Tr2 DT during larval stages. Shown in the figures are H 2 DCFDA staining in L2 ( B ), 0–8 hr L3 ( C ), 16–24 hr L3 ( D ), and 32–40 hr L3 ( E ) in wild type ( btl-Gal4 ) animals. ( F–I ) Levels of the ROS reporter dihydroethidium (DHE) in Tr2 DT during larval stages. Shown in the figures are DHE staining in L2 ( F ), 0–8 hr L3 ( G ), 16–24 hr L3 ( H ), and 32–40 hr L3 ( I ) in wild type ( btl-Gal4 ) animals. ( J, K ) Effect of btl -Gal4-dependent overexpression of superoxide dismutase 1 (SOD1) on levels of ROS reporters in Tr2 DT. ( J ) H 2 DCFDA staining in btl-SOD1 ( btl-GAL4/UAS-SOD1 )-expressing larvae (n ≥ 6 tracheae per condition per timepoint). ( K ) DHE staining in btl-SOD1 larvae (n ≥ 6 tracheae per condition per timepoint). ( L ) Effect of SOD1 overexpression on cell numbers in Tr2 DT at different larval stages. Graph shows numbers of Tr2 tracheoblasts in wild type ( btl-Gal4), btl-SOD1 (btl-GAL4/UAS-SOD1 ), and <t>btl-Chk1</t> RNAi (btl-GAL4/UAS-Chk1 RNAi ) larvae at L2, 0–8 hr L3, 16–24 hr L3, 32–40 hr L3, and wandering L3 (WL3) (n ≥ 7 tracheae per condition per timepoint). ( M ) Effect of SOD1 overexpression on mitotic indices in Tr2 DT (see text). Graph shows mitotic indices in Tr2 DT in wild type and btl-SOD1 (btl-GAL4/UAS-SOD1 )-expressing larvae at L2, 0–8 hr L3 and 16–24 hr L3 (mean values ± standard deviation, n ≥ 7 tracheae per condition per timepoint). ( N ) Effect of SOD1 overexpression on Chk1 phosphorylation in Tr2 tracheoblasts. Shown in the figure is phosphorylated Chk1 (pChk1, phospho-Chk1Ser 345 ) immunostaining (red) in Tr2 DT in wild type ( btl-GAL4 ) and btl-SOD1 (btl-GAL4/UAS-SOD1 ) larvae at L2. Scale bars = 10 µm. Dashed lines outline the cuticular lumen of the tracheal tube here and elsewhere and are shifted outward to include the epithelial lining when they overlap with the signal. Student’s t-test: *p<0.00001. Figure 1—source data 1. Cell Frequencies in SOD1 overexpressing animals. Figure 1—source data 2. Mitotic indices in SOD1 overexpressing animals.
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    The ability of NVP-AUY922 to abrogate G 2 arrest in the presence of dsDNA damage was investigated by western blot and FACS analysis in all four cell lines (p53 status: HCT116 p53wt, Hela HPV-positive, HN5 and HN3 p53 mutant). (A) HeLa and HN3 cells were pre-treated with NVP-AUY922 for 16 h at the concentrations indicated. Cells were then mock irradiated or irradiated with 4 Gy and 4 h later whole cell lysates harvested, 30 µg protein resolved on 10% SDS-PAGE then probed for phospho-H2ax, phospho-histone H3 and phospho-CHK1 by western blot. (B) The effect of NVP-AUY922 on depletion of total-CHK1 was also investigated in unirradiated cells with γ-tubulin probed as loading control. (C+D) Cells were exposed to the NVP-AUY922 concentrations indicated for 16 h before mock irradiation or irradiation with 4 Gy. Cells were fixed 9 h and 48 h post-irradiation before staining for the mitotic marker p-histone H3 and DNA content with propidium iodide. SubG 1 and >4N populations were quantified by FACS analysis. Data represents ± SEM of three independent experiments each recording at least 10,000 events. Statistical analysis carried out by two-tailed t-test between the groups indicated, *p<0.05 **p<0.01 ***p<0.001.

    Journal: PLoS ONE

    Article Title: The HSP90 Inhibitor NVP-AUY922 Radiosensitizes by Abrogation of Homologous Recombination Resulting in Mitotic Entry with Unresolved DNA Damage

    doi: 10.1371/journal.pone.0035436

    Figure Lengend Snippet: The ability of NVP-AUY922 to abrogate G 2 arrest in the presence of dsDNA damage was investigated by western blot and FACS analysis in all four cell lines (p53 status: HCT116 p53wt, Hela HPV-positive, HN5 and HN3 p53 mutant). (A) HeLa and HN3 cells were pre-treated with NVP-AUY922 for 16 h at the concentrations indicated. Cells were then mock irradiated or irradiated with 4 Gy and 4 h later whole cell lysates harvested, 30 µg protein resolved on 10% SDS-PAGE then probed for phospho-H2ax, phospho-histone H3 and phospho-CHK1 by western blot. (B) The effect of NVP-AUY922 on depletion of total-CHK1 was also investigated in unirradiated cells with γ-tubulin probed as loading control. (C+D) Cells were exposed to the NVP-AUY922 concentrations indicated for 16 h before mock irradiation or irradiation with 4 Gy. Cells were fixed 9 h and 48 h post-irradiation before staining for the mitotic marker p-histone H3 and DNA content with propidium iodide. SubG 1 and >4N populations were quantified by FACS analysis. Data represents ± SEM of three independent experiments each recording at least 10,000 events. Statistical analysis carried out by two-tailed t-test between the groups indicated, *p<0.05 **p<0.01 ***p<0.001.

    Article Snippet: 20–30 µg total protein were separated by reducing 10% SDS-PAGE, transferred to PVDF membrane (GE Healthcare, Bucks, UK), blocked with 5% non-fat dry milk in PBS for 1 h and probed with the following primary antibodies in 5% BSA and 0.1% Tween-20: rabbit anti-HSP72 from Stressgen (Exeter, UK), rabbit anti-ErbB2, rabbit anti-cRAF from Santacruz (CA, USA), rabbit anti-pErbB2, rabbit anti-pCHK1, rabbit anti-CHK1, rabbit anti-pAKT, rabbit anti-AKT and rabbit anti-pH2ax were all purchased from Cell Signalling (MA, USA), rabbit anti-p-histone H3 was purchased from Upstate-Millipore (UK).

    Techniques: Western Blot, Mutagenesis, Irradiation, SDS Page, Staining, Marker, Two Tailed Test

    Unsynchronized HeLa cells were treated with 0, 20, or 30 µM DhL and lysed or fixed at the indicated times. (A) Immunoblot analysis of phospho-ATM (p-ATM) and phospho-Chk1 (p-Chk1). Representative assay of 3 independent experiments. β-actin was employed as a loading control. Cells were stained with DAPI to visualize the nuclei and treated with specific antibodies for γH2AX (B) and 53BP1 (C). Left: representative fields from 24 h treatment. Insets: magnification of the areas indicated by boxes in the top row. Representative fields for 48 h treatment are shown in . Right: quantification of the number of cells with more than 10 γH2AX foci and more than 5 53BP1 foci. At least 200 nuclei were scored for each sample. Bar: 10 µm. Data represent mean ± SEM of 3 independent experiments. * p≤0.05, ** p≤0.01 vs. control group (0 µM DhL).

    Journal: PLoS ONE

    Article Title: The Sesquiterpene Lactone Dehydroleucodine Triggers Senescence and Apoptosis in Association with Accumulation of DNA Damage Markers

    doi: 10.1371/journal.pone.0053168

    Figure Lengend Snippet: Unsynchronized HeLa cells were treated with 0, 20, or 30 µM DhL and lysed or fixed at the indicated times. (A) Immunoblot analysis of phospho-ATM (p-ATM) and phospho-Chk1 (p-Chk1). Representative assay of 3 independent experiments. β-actin was employed as a loading control. Cells were stained with DAPI to visualize the nuclei and treated with specific antibodies for γH2AX (B) and 53BP1 (C). Left: representative fields from 24 h treatment. Insets: magnification of the areas indicated by boxes in the top row. Representative fields for 48 h treatment are shown in . Right: quantification of the number of cells with more than 10 γH2AX foci and more than 5 53BP1 foci. At least 200 nuclei were scored for each sample. Bar: 10 µm. Data represent mean ± SEM of 3 independent experiments. * p≤0.05, ** p≤0.01 vs. control group (0 µM DhL).

    Article Snippet: Antibodies against phospho-Histone H2AX, phospho-Histone H3 and phospho ATM were from Millipore, (California), anti-phospho Chk1 wasfrom Cell Signaling Technology, anti p53 (1801 and DO1) were gifts from Carol Prives-Columbia University New York and anti p21 was from Santa Cruz Biotechnology.

    Techniques: Western Blot, Staining

    (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of pCHK1/CHK1 and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.

    Journal: PLoS ONE

    Article Title: Mutation of Serine 1333 in the ATR HEAT Repeats Creates a Hyperactive Kinase

    doi: 10.1371/journal.pone.0099397

    Figure Lengend Snippet: (A) Lysates from ATR−/− cell clones expressing wild type (W1, W5, W7), S1333A (A2, A3, A6), or S1333D (D2, D5, D7) ATR proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Quantitative immunoblotting was used and the ratio of phosphorylated protein to total protein normalized to wild type (W1) is listed below each lane. Note that three clonal isolates for each ATR protein were analyzed to ensure results were not due to clonal variation. All cell lines were examined multiple times and a representative experiment is shown. (B) The ratio of pCHK1/CHK1 and the expression levels of ATR are compared to show that the small differences in ATR expression levels in different cell lines do not account for the change in substrate phosphorylation.

    Article Snippet: Antibodies used include ATR-N19 (Santa Cruz Biotechnology), HA (Covance), CHK1-G4 (Santa Cruz Biotechnology), FLAG-M2 (Sigma), ATRIP403 , MCM2 (BD Transduction Labs), phosphorylated Ser-317 CHK1 (Cell Signaling Technology), phosphorylated Ser-345 CHK1 (Cell Signaling Technology), and phosphorylated Ser-10 Histone H3 (Cell Signaling Technology).

    Techniques: Clone Assay, Expressing, SDS Page, Western Blot

    Mcph1 -Δe8 cells exhibit PCC and defective DDR. ( A ) Representative images of PCC in Mcph1 -Δe8 MEFs. Primary MEF cells were stained with a pS10-H3 antibody (red) and counterstained with DAPI (blue). ( B ) Quantification of the percentages of PCC cells (prophase cells lacking the pS10-H3 signal) in control and Mcph1 -Δe8 primary MEF cells. More than 250 prophase cells were scored in each group of the indicated genotype. ( C ) Western blot analysis of p-Chk1 and γH2AX in control and Mcph1 -Δe8 MEFs with or without HU treatment. β-actin was used as a loading control. ( D ) The quantification of the indicated protein intensities from panel ( C ). Unpaired Student’s t -test was used for statistical analysis. ***, p < 0.001.

    Journal: Cells

    Article Title: The Central Domain of MCPH1 Controls Development of the Cerebral Cortex and Gonads in Mice

    doi: 10.3390/cells11172715

    Figure Lengend Snippet: Mcph1 -Δe8 cells exhibit PCC and defective DDR. ( A ) Representative images of PCC in Mcph1 -Δe8 MEFs. Primary MEF cells were stained with a pS10-H3 antibody (red) and counterstained with DAPI (blue). ( B ) Quantification of the percentages of PCC cells (prophase cells lacking the pS10-H3 signal) in control and Mcph1 -Δe8 primary MEF cells. More than 250 prophase cells were scored in each group of the indicated genotype. ( C ) Western blot analysis of p-Chk1 and γH2AX in control and Mcph1 -Δe8 MEFs with or without HU treatment. β-actin was used as a loading control. ( D ) The quantification of the indicated protein intensities from panel ( C ). Unpaired Student’s t -test was used for statistical analysis. ***, p < 0.001.

    Article Snippet: In order to investigate the DNA repair dynamics, the following antibodies were used: mouse anti-phospho histone H2A.X (Ser139) (1:2000, 05-636, EMD Millipore, Burlington, MA, USA), rabbit anti-phospho Chk1 (Ser317) (1:1000,12302S, Cell Signaling, Danvers, MA, USA) and mouse anti-β-actin (1:10,000, A5441, Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Staining, Western Blot

    Irreparable damage is not channeled for apoptosis due to timing defects in ATM/ATR signaling. ( A ) Quantification of the number of pachytene nuclei with CED-1::GFP engulfment. Each data point reflects the number of nuclei in one gonad completely engulfed by CED-1::GFP. Black lines indicate median. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). ( B ) Quantification of the number of nuclei with acridine orange staining in the indicated backgrounds. Each data point reflects the number of acridine orange-positive nuclei in one gonad. Statistical significance calculated by Mann–Whitney t -test. ( C ) Quantification of the number of late pachytene nuclei with (+) or without (−) PGL-1 staining. Each data point reflects the number of nuclei within one gonad. Error bars reflect mean with SEM. Statistical comparisons drawn between quantifications of the same category (e.g. PGL-1(+) v. PGL-1(+)) by Mann–Whitney t-test. ( D ) Quantification of the number of FLAG::RPA-2 foci across the germline. X-axis denotes the zone of the germline and the y-axis reflects the average number of FLAG foci. Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). ( E ) Measurements of ATM/ATR signaling. Fluorescence intensity of (pS/pT) QG signal across the gonad. X-axis denotes the zone of the germline and the y-axis denotes arbitrary fluorescent units per μm 2 . Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). Gray asterisks reflect significance compared to wild type, purple asterisks to syp-3 , and pink asterisks to rnh-1.0; rnh-2 . ( F ) Quantification of the number of CHK-1 foci in late pachytene nuclei. Error bars reflect mean with SEM. Statistical significance calculated by Mann–Whitney t -test. ( G ) Fluorescence intensity of CEP-1::GFP signal in late pachytene nuclei. Each data point reflects the fluorescence intensity of one LP nucleus with correction to the average background signal for that gonad. Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05).

    Journal: Nucleic Acids Research

    Article Title: R-loop-induced irreparable DNA damage evades checkpoint detection in the C. elegans germline

    doi: 10.1093/nar/gkac621

    Figure Lengend Snippet: Irreparable damage is not channeled for apoptosis due to timing defects in ATM/ATR signaling. ( A ) Quantification of the number of pachytene nuclei with CED-1::GFP engulfment. Each data point reflects the number of nuclei in one gonad completely engulfed by CED-1::GFP. Black lines indicate median. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). ( B ) Quantification of the number of nuclei with acridine orange staining in the indicated backgrounds. Each data point reflects the number of acridine orange-positive nuclei in one gonad. Statistical significance calculated by Mann–Whitney t -test. ( C ) Quantification of the number of late pachytene nuclei with (+) or without (−) PGL-1 staining. Each data point reflects the number of nuclei within one gonad. Error bars reflect mean with SEM. Statistical comparisons drawn between quantifications of the same category (e.g. PGL-1(+) v. PGL-1(+)) by Mann–Whitney t-test. ( D ) Quantification of the number of FLAG::RPA-2 foci across the germline. X-axis denotes the zone of the germline and the y-axis reflects the average number of FLAG foci. Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). ( E ) Measurements of ATM/ATR signaling. Fluorescence intensity of (pS/pT) QG signal across the gonad. X-axis denotes the zone of the germline and the y-axis denotes arbitrary fluorescent units per μm 2 . Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05). Gray asterisks reflect significance compared to wild type, purple asterisks to syp-3 , and pink asterisks to rnh-1.0; rnh-2 . ( F ) Quantification of the number of CHK-1 foci in late pachytene nuclei. Error bars reflect mean with SEM. Statistical significance calculated by Mann–Whitney t -test. ( G ) Fluorescence intensity of CEP-1::GFP signal in late pachytene nuclei. Each data point reflects the fluorescence intensity of one LP nucleus with correction to the average background signal for that gonad. Error bars reflect mean with SEM. Asterisks indicate statistical significance calculated by Mann–Whitney t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05).

    Article Snippet: Rabbit anti-CHK-1 (Cell Signaling 4539, 1:100), AlexaFluor 488 α-rabbit (Molecular Probes/Invitrogen, A32790, 1:500), and mouse anti-fibrillarin (72B9, gift from P. DiMario).

    Techniques: MANN-WHITNEY, Staining, Fluorescence

    The control rad9A +/+ (+/+) and rad9A -/- (KO) PC3 cells transfected with vector (V), FLAG-WT-hRad9A (WT), FLAG-DM-hRad9A (DM), FLAG-LD-hRad9A (LD) or FLAG-K220A-hRad9A (K220A) were treated with 16 mM hydroxyurea (HU) for 2 hours and recovered for 2 hours or left untreated. Cell extracts were subjected to Western blotting analysis with antibodies against phosphorylated Chk1 (pChk1) at Ser317 (S-317) ( A ), total Chk1 ( B ), and β-actin ( C ). Molecular weight markers were loaded on lane 1 with indicated sizes in KDa.

    Journal: PLoS ONE

    Article Title: DNA binding by the Rad9A subunit of the Rad9-Rad1-Hus1 complex

    doi: 10.1371/journal.pone.0272645

    Figure Lengend Snippet: The control rad9A +/+ (+/+) and rad9A -/- (KO) PC3 cells transfected with vector (V), FLAG-WT-hRad9A (WT), FLAG-DM-hRad9A (DM), FLAG-LD-hRad9A (LD) or FLAG-K220A-hRad9A (K220A) were treated with 16 mM hydroxyurea (HU) for 2 hours and recovered for 2 hours or left untreated. Cell extracts were subjected to Western blotting analysis with antibodies against phosphorylated Chk1 (pChk1) at Ser317 (S-317) ( A ), total Chk1 ( B ), and β-actin ( C ). Molecular weight markers were loaded on lane 1 with indicated sizes in KDa.

    Article Snippet: The membranes were allowed to react with antibodies against phosphorylated Chk1 (pChk1) at Ser317 (S317) (Cell Signaling), total Chk1 (Bethyl Laboratories) or β-actin (Sigma).

    Techniques: Transfection, Plasmid Preparation, Western Blot, Molecular Weight

    ( A ) A diagram of the third instar larva showing the dorsal trunk (DT) of the second thoracic metamere (Tr2, colored in green and marked by dashed line). The diagram also shows the timecourse of G2 arrest and cell division in Tr2. The cells in Tr2 DT remain geographically isolated from tracheal cells in other branches during larval life. ( B–E ) Levels of the ROS reporter 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCFDA) in Tr2 DT during larval stages. Shown in the figures are H 2 DCFDA staining in L2 ( B ), 0–8 hr L3 ( C ), 16–24 hr L3 ( D ), and 32–40 hr L3 ( E ) in wild type ( btl-Gal4 ) animals. ( F–I ) Levels of the ROS reporter dihydroethidium (DHE) in Tr2 DT during larval stages. Shown in the figures are DHE staining in L2 ( F ), 0–8 hr L3 ( G ), 16–24 hr L3 ( H ), and 32–40 hr L3 ( I ) in wild type ( btl-Gal4 ) animals. ( J, K ) Effect of btl -Gal4-dependent overexpression of superoxide dismutase 1 (SOD1) on levels of ROS reporters in Tr2 DT. ( J ) H 2 DCFDA staining in btl-SOD1 ( btl-GAL4/UAS-SOD1 )-expressing larvae (n ≥ 6 tracheae per condition per timepoint). ( K ) DHE staining in btl-SOD1 larvae (n ≥ 6 tracheae per condition per timepoint). ( L ) Effect of SOD1 overexpression on cell numbers in Tr2 DT at different larval stages. Graph shows numbers of Tr2 tracheoblasts in wild type ( btl-Gal4), btl-SOD1 (btl-GAL4/UAS-SOD1 ), and btl-Chk1 RNAi (btl-GAL4/UAS-Chk1 RNAi ) larvae at L2, 0–8 hr L3, 16–24 hr L3, 32–40 hr L3, and wandering L3 (WL3) (n ≥ 7 tracheae per condition per timepoint). ( M ) Effect of SOD1 overexpression on mitotic indices in Tr2 DT (see text). Graph shows mitotic indices in Tr2 DT in wild type and btl-SOD1 (btl-GAL4/UAS-SOD1 )-expressing larvae at L2, 0–8 hr L3 and 16–24 hr L3 (mean values ± standard deviation, n ≥ 7 tracheae per condition per timepoint). ( N ) Effect of SOD1 overexpression on Chk1 phosphorylation in Tr2 tracheoblasts. Shown in the figure is phosphorylated Chk1 (pChk1, phospho-Chk1Ser 345 ) immunostaining (red) in Tr2 DT in wild type ( btl-GAL4 ) and btl-SOD1 (btl-GAL4/UAS-SOD1 ) larvae at L2. Scale bars = 10 µm. Dashed lines outline the cuticular lumen of the tracheal tube here and elsewhere and are shifted outward to include the epithelial lining when they overlap with the signal. Student’s t-test: *p<0.00001. Figure 1—source data 1. Cell Frequencies in SOD1 overexpressing animals. Figure 1—source data 2. Mitotic indices in SOD1 overexpressing animals.

    Journal: eLife

    Article Title: Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts

    doi: 10.7554/eLife.68636

    Figure Lengend Snippet: ( A ) A diagram of the third instar larva showing the dorsal trunk (DT) of the second thoracic metamere (Tr2, colored in green and marked by dashed line). The diagram also shows the timecourse of G2 arrest and cell division in Tr2. The cells in Tr2 DT remain geographically isolated from tracheal cells in other branches during larval life. ( B–E ) Levels of the ROS reporter 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCFDA) in Tr2 DT during larval stages. Shown in the figures are H 2 DCFDA staining in L2 ( B ), 0–8 hr L3 ( C ), 16–24 hr L3 ( D ), and 32–40 hr L3 ( E ) in wild type ( btl-Gal4 ) animals. ( F–I ) Levels of the ROS reporter dihydroethidium (DHE) in Tr2 DT during larval stages. Shown in the figures are DHE staining in L2 ( F ), 0–8 hr L3 ( G ), 16–24 hr L3 ( H ), and 32–40 hr L3 ( I ) in wild type ( btl-Gal4 ) animals. ( J, K ) Effect of btl -Gal4-dependent overexpression of superoxide dismutase 1 (SOD1) on levels of ROS reporters in Tr2 DT. ( J ) H 2 DCFDA staining in btl-SOD1 ( btl-GAL4/UAS-SOD1 )-expressing larvae (n ≥ 6 tracheae per condition per timepoint). ( K ) DHE staining in btl-SOD1 larvae (n ≥ 6 tracheae per condition per timepoint). ( L ) Effect of SOD1 overexpression on cell numbers in Tr2 DT at different larval stages. Graph shows numbers of Tr2 tracheoblasts in wild type ( btl-Gal4), btl-SOD1 (btl-GAL4/UAS-SOD1 ), and btl-Chk1 RNAi (btl-GAL4/UAS-Chk1 RNAi ) larvae at L2, 0–8 hr L3, 16–24 hr L3, 32–40 hr L3, and wandering L3 (WL3) (n ≥ 7 tracheae per condition per timepoint). ( M ) Effect of SOD1 overexpression on mitotic indices in Tr2 DT (see text). Graph shows mitotic indices in Tr2 DT in wild type and btl-SOD1 (btl-GAL4/UAS-SOD1 )-expressing larvae at L2, 0–8 hr L3 and 16–24 hr L3 (mean values ± standard deviation, n ≥ 7 tracheae per condition per timepoint). ( N ) Effect of SOD1 overexpression on Chk1 phosphorylation in Tr2 tracheoblasts. Shown in the figure is phosphorylated Chk1 (pChk1, phospho-Chk1Ser 345 ) immunostaining (red) in Tr2 DT in wild type ( btl-GAL4 ) and btl-SOD1 (btl-GAL4/UAS-SOD1 ) larvae at L2. Scale bars = 10 µm. Dashed lines outline the cuticular lumen of the tracheal tube here and elsewhere and are shifted outward to include the epithelial lining when they overlap with the signal. Student’s t-test: *p<0.00001. Figure 1—source data 1. Cell Frequencies in SOD1 overexpressing animals. Figure 1—source data 2. Mitotic indices in SOD1 overexpressing animals.

    Article Snippet: The following antisera were used for immunohistochemical analysis: chicken anti-GFP (Aves, 1:500, RRID: AB_10000240 ), rabbit anti-phospho Chk1 (CST, 1:200, RRID: AB_331212 ), rabbit anti-pH3 (Millipore, 1:500, RRID: AB_310177 ), mouse anti-8-hydroxy-2′-deoxyguanosine (Abcam, 1:200, RRID: AB_867461 ), and Alexa 488/568-conjugated donkey anti-chicken/rabbit/mouse secondary antibodies (Invitrogen, 1:200).

    Techniques: Isolation, Staining, Over Expression, Expressing, Standard Deviation, Immunostaining

    ( A–D ) Detailed analysis of DNA damage in Tr2 dorsal trunk (DT). Shown here are findings from three different reporters of genotoxic stress. ( A ) 8-Oxo-2'-deoxyguanosine (8-Oxo-dG) immunostaining in wild type ( btl-GAL4 ) Tr2 DT in untreated tracheae (left panel) and tracheae exposed to 1 mM H 2 O 2 for 30 min ex vivo (right panel) at L2. ( B ) 8-Oxo-dG immunostaining in wild type ( btl-GAL4 ) endocycling cells of the tracheae exposed to 1 mM H 2 O 2 for 30 min ex vivo at L2. ( C ) GFP immunostaining in non-irradiated and γ-irradiated larvae expressing RPA70-GFP. Shown in the figure are GFP immunostaining in non-irradiated larvae (top panel) and larvae exposed to 50 Gy of γ-radiation (bottom panel) at L2. ( D ) γ-H2AX Ser139 immunostaining in Tr2 DT in wild type ( btl-GAL4 ) non-irradiated larvae (top panel) and larvae irradiated with 50 Gy of γ-radiation (bottom panel) at L2. ( E–H ) Analysis of the contribution of components of the DNA damage-dependent activation of ATR/Chk1 to Chk1 activation in Tr2 DT. Effects of the knockdown of ATR, ATRIP, TOPBP1, and Claspin on phosphorylated checkpoint kinase 1 (pChk1) levels in Tr2 DT at L2. pChk1 immunostaining (red) in Tr2 DT in ( E ) btl-ATR RNAi ( btl-GAL4/UAS-ATR RNAi ), ( F ) btl-ATRIP RNAi ( btl-GAL4/UAS-ATRIP RNAi ), ( G ) btl-TOPBP1 RNAi ( btl-GAL4/+; UAS-TOPBP1 RNAi /+ ), and ( H ) btl-Claspin RNAi ( btl-GAL4/+; UAS-Claspin RNAi /+ ) larvae at L2. ( I ) Effects of knockdown of ATR, ATRIP, TOPBP1, and Claspin on cell numbers in Tr2 DT. Graph shows numbers of Tr2 tracheoblasts in wild type ( btl-Gal4 ), btl-ATR RNAi ( btl-GAL4/UAS-ATR RNAi ), btl-ATRIP RNAi ( btl-GAL4/UAS-ATRIP RNAi ), btl-TOPBP1 RNAi ( btl-GAL4/+; UAS-TOPBP1 RNAi /+ ), and btl-Claspin RNAi ( btl-GAL4/+; UAS-Claspin RNAi /+ ) at L2 and 16–24 hr L3 (mean values ± standard deviation, n ≥ 7 tracheae per condition per timepoint). Scale bars = 5 µm ( A–D ), 10 µm ( E–H ). Student’s t-test: *p<0.00001. Figure 4—source data 1. Cell frequencies in ATR RNAi , ATRIP RNAi , TOPBP1 RNAi and Claspin RNAi expressing animals.

    Journal: eLife

    Article Title: Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts

    doi: 10.7554/eLife.68636

    Figure Lengend Snippet: ( A–D ) Detailed analysis of DNA damage in Tr2 dorsal trunk (DT). Shown here are findings from three different reporters of genotoxic stress. ( A ) 8-Oxo-2'-deoxyguanosine (8-Oxo-dG) immunostaining in wild type ( btl-GAL4 ) Tr2 DT in untreated tracheae (left panel) and tracheae exposed to 1 mM H 2 O 2 for 30 min ex vivo (right panel) at L2. ( B ) 8-Oxo-dG immunostaining in wild type ( btl-GAL4 ) endocycling cells of the tracheae exposed to 1 mM H 2 O 2 for 30 min ex vivo at L2. ( C ) GFP immunostaining in non-irradiated and γ-irradiated larvae expressing RPA70-GFP. Shown in the figure are GFP immunostaining in non-irradiated larvae (top panel) and larvae exposed to 50 Gy of γ-radiation (bottom panel) at L2. ( D ) γ-H2AX Ser139 immunostaining in Tr2 DT in wild type ( btl-GAL4 ) non-irradiated larvae (top panel) and larvae irradiated with 50 Gy of γ-radiation (bottom panel) at L2. ( E–H ) Analysis of the contribution of components of the DNA damage-dependent activation of ATR/Chk1 to Chk1 activation in Tr2 DT. Effects of the knockdown of ATR, ATRIP, TOPBP1, and Claspin on phosphorylated checkpoint kinase 1 (pChk1) levels in Tr2 DT at L2. pChk1 immunostaining (red) in Tr2 DT in ( E ) btl-ATR RNAi ( btl-GAL4/UAS-ATR RNAi ), ( F ) btl-ATRIP RNAi ( btl-GAL4/UAS-ATRIP RNAi ), ( G ) btl-TOPBP1 RNAi ( btl-GAL4/+; UAS-TOPBP1 RNAi /+ ), and ( H ) btl-Claspin RNAi ( btl-GAL4/+; UAS-Claspin RNAi /+ ) larvae at L2. ( I ) Effects of knockdown of ATR, ATRIP, TOPBP1, and Claspin on cell numbers in Tr2 DT. Graph shows numbers of Tr2 tracheoblasts in wild type ( btl-Gal4 ), btl-ATR RNAi ( btl-GAL4/UAS-ATR RNAi ), btl-ATRIP RNAi ( btl-GAL4/UAS-ATRIP RNAi ), btl-TOPBP1 RNAi ( btl-GAL4/+; UAS-TOPBP1 RNAi /+ ), and btl-Claspin RNAi ( btl-GAL4/+; UAS-Claspin RNAi /+ ) at L2 and 16–24 hr L3 (mean values ± standard deviation, n ≥ 7 tracheae per condition per timepoint). Scale bars = 5 µm ( A–D ), 10 µm ( E–H ). Student’s t-test: *p<0.00001. Figure 4—source data 1. Cell frequencies in ATR RNAi , ATRIP RNAi , TOPBP1 RNAi and Claspin RNAi expressing animals.

    Article Snippet: The following antisera were used for immunohistochemical analysis: chicken anti-GFP (Aves, 1:500, RRID: AB_10000240 ), rabbit anti-phospho Chk1 (CST, 1:200, RRID: AB_331212 ), rabbit anti-pH3 (Millipore, 1:500, RRID: AB_310177 ), mouse anti-8-hydroxy-2′-deoxyguanosine (Abcam, 1:200, RRID: AB_867461 ), and Alexa 488/568-conjugated donkey anti-chicken/rabbit/mouse secondary antibodies (Invitrogen, 1:200).

    Techniques: Immunostaining, Ex Vivo, Irradiation, Expressing, Activation Assay, Standard Deviation

    Effect of knockdown of Duox and ATRIP or TOPBP1 or Claspin on phosphorylated checkpoint kinase 1 (pChk1) levels in Tr2 dorsal trunk (DT) in larvae exposed to 50 Gy of γ-radiation at L2. ( A ) Schematic describing the protocol for exposure to γ-radiation and immunostaining. ( B–D ) pChk1 immunostaining in Tr2 DT in ( B ) btl-Duox RNAi , ATRIP RNAi (btl-GAL4/UAS-ATRIP RNAi ; UAS-Duox RNAi (32903)/+ ), ( C ) btl-Duox RNAi , TOPBP1 RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-TOPBP1 RNAi ), and ( D ) btl-Duox RNAi , Claspin RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-Claspin RNAi ) larvae at 1 hr post exposure to 50 Gy of γ-radiation at L2 (n ≥ 6 tracheae per condition). Scale bars = 10 µm.

    Journal: eLife

    Article Title: Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts

    doi: 10.7554/eLife.68636

    Figure Lengend Snippet: Effect of knockdown of Duox and ATRIP or TOPBP1 or Claspin on phosphorylated checkpoint kinase 1 (pChk1) levels in Tr2 dorsal trunk (DT) in larvae exposed to 50 Gy of γ-radiation at L2. ( A ) Schematic describing the protocol for exposure to γ-radiation and immunostaining. ( B–D ) pChk1 immunostaining in Tr2 DT in ( B ) btl-Duox RNAi , ATRIP RNAi (btl-GAL4/UAS-ATRIP RNAi ; UAS-Duox RNAi (32903)/+ ), ( C ) btl-Duox RNAi , TOPBP1 RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-TOPBP1 RNAi ), and ( D ) btl-Duox RNAi , Claspin RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-Claspin RNAi ) larvae at 1 hr post exposure to 50 Gy of γ-radiation at L2 (n ≥ 6 tracheae per condition). Scale bars = 10 µm.

    Article Snippet: The following antisera were used for immunohistochemical analysis: chicken anti-GFP (Aves, 1:500, RRID: AB_10000240 ), rabbit anti-phospho Chk1 (CST, 1:200, RRID: AB_331212 ), rabbit anti-pH3 (Millipore, 1:500, RRID: AB_310177 ), mouse anti-8-hydroxy-2′-deoxyguanosine (Abcam, 1:200, RRID: AB_867461 ), and Alexa 488/568-conjugated donkey anti-chicken/rabbit/mouse secondary antibodies (Invitrogen, 1:200).

    Techniques: Immunostaining

    ( A–E ) Kinetics of Chk1 phosphorylation on exposure to γ-radiation. ( A ) Regimen for γ-irradiation and analysis of pChk1. pChk1 immunostaining (red) in Tr2 dorsal trunk (DT) in btl-Duox RNAi (btl-GAL4/+; UAS-Duox RNAi (32903/+ )) in non-irradiated larvae ( B ) and larvae exposed to with 50 Gy of γ-radiation after ( C ) 1 hr, ( D ) 30 min, and ( E ) 2 min post irradiation at L2. ( F ) Effect of knockdown of ATR on Chk1 activation in Tr2 DT in larvae exposed to γ-radiation. pChk1 immunostaining (red) in Tr2 DT in btl-ATR RNAi (btl-GAL4/UAS-ATR RNAi ) larvae exposed to 50 Gy of γ-radiation at 1 hr post exposure at L2 (n ≥ 6 tracheae per condition) Scale bars = 10 µm.

    Journal: eLife

    Article Title: Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts

    doi: 10.7554/eLife.68636

    Figure Lengend Snippet: ( A–E ) Kinetics of Chk1 phosphorylation on exposure to γ-radiation. ( A ) Regimen for γ-irradiation and analysis of pChk1. pChk1 immunostaining (red) in Tr2 dorsal trunk (DT) in btl-Duox RNAi (btl-GAL4/+; UAS-Duox RNAi (32903/+ )) in non-irradiated larvae ( B ) and larvae exposed to with 50 Gy of γ-radiation after ( C ) 1 hr, ( D ) 30 min, and ( E ) 2 min post irradiation at L2. ( F ) Effect of knockdown of ATR on Chk1 activation in Tr2 DT in larvae exposed to γ-radiation. pChk1 immunostaining (red) in Tr2 DT in btl-ATR RNAi (btl-GAL4/UAS-ATR RNAi ) larvae exposed to 50 Gy of γ-radiation at 1 hr post exposure at L2 (n ≥ 6 tracheae per condition) Scale bars = 10 µm.

    Article Snippet: The following antisera were used for immunohistochemical analysis: chicken anti-GFP (Aves, 1:500, RRID: AB_10000240 ), rabbit anti-phospho Chk1 (CST, 1:200, RRID: AB_331212 ), rabbit anti-pH3 (Millipore, 1:500, RRID: AB_310177 ), mouse anti-8-hydroxy-2′-deoxyguanosine (Abcam, 1:200, RRID: AB_867461 ), and Alexa 488/568-conjugated donkey anti-chicken/rabbit/mouse secondary antibodies (Invitrogen, 1:200).

    Techniques: Irradiation, Immunostaining, Activation Assay

    ( A–E ) Kinetics of Chk1 phosphorylation upon exposure to H 2 O 2 ex vivo. ( A ) Regimen for H 2 O 2 treatment and analysis of pChk1 in Tr2 dorsal trunk (DT) in L2. pChk1 immunostaining (red) in Tr2 DT in ( B ) untreated btl-Duox RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/+ )-expressing tracheae and treated with 100 µM H 2 O 2 for ( C ) 30 min, ( D ) 5 min, and ( E ) 2 min. ( F ) Effect of knockdown of ATR on Chk1 activation in Tr2 DT upon exposure to H 2 O 2 ex vivo. pChk1 immunostaining (red) in Tr2 DT in btl-ATR RNAi (btl-GAL4/UAS-ATR RNAi ) tracheae treated with 100 µM H 2 O 2 for 30 min. ( G–I ) Effect of knockdown of Duox and ATRIP or TOPBP1 or Claspin on pChk1 levels in Tr2 DT in tracheae exposed to 100 µM H 2 O 2 at L2. pChk1 immunostaining (red) in Tr2 DT in ( G ) btl-Duox RNAi , ATRIP RNAi ( btl-GAL4/ UAS-ATRIP RNAi ; UAS-Duox RNAi (32903)/+ ), ( H ) btl -Duox RNAi , TOPBP1 RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-TOPBP1 RNAi ) and ( I ) btl-Duox RNAi , Claspin RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-Claspin RNAi ) tracheae treated with 100 µM H 2 O 2 for 2 min at L2. ( J ) Model for the regulation of ATR/Chk1 activation in Tr2 DT. We propose that H 2 O 2 can induce ATR-dependent phosphorylation and activation of Chk1 in the absence of detectable DNA damage, leading to G2 arrest in Tr2 tracheoblasts. Scale bars = 10 µm.

    Journal: eLife

    Article Title: Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts

    doi: 10.7554/eLife.68636

    Figure Lengend Snippet: ( A–E ) Kinetics of Chk1 phosphorylation upon exposure to H 2 O 2 ex vivo. ( A ) Regimen for H 2 O 2 treatment and analysis of pChk1 in Tr2 dorsal trunk (DT) in L2. pChk1 immunostaining (red) in Tr2 DT in ( B ) untreated btl-Duox RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/+ )-expressing tracheae and treated with 100 µM H 2 O 2 for ( C ) 30 min, ( D ) 5 min, and ( E ) 2 min. ( F ) Effect of knockdown of ATR on Chk1 activation in Tr2 DT upon exposure to H 2 O 2 ex vivo. pChk1 immunostaining (red) in Tr2 DT in btl-ATR RNAi (btl-GAL4/UAS-ATR RNAi ) tracheae treated with 100 µM H 2 O 2 for 30 min. ( G–I ) Effect of knockdown of Duox and ATRIP or TOPBP1 or Claspin on pChk1 levels in Tr2 DT in tracheae exposed to 100 µM H 2 O 2 at L2. pChk1 immunostaining (red) in Tr2 DT in ( G ) btl-Duox RNAi , ATRIP RNAi ( btl-GAL4/ UAS-ATRIP RNAi ; UAS-Duox RNAi (32903)/+ ), ( H ) btl -Duox RNAi , TOPBP1 RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-TOPBP1 RNAi ) and ( I ) btl-Duox RNAi , Claspin RNAi (btl-GAL4/+; UAS-Duox RNAi (32903)/UAS-Claspin RNAi ) tracheae treated with 100 µM H 2 O 2 for 2 min at L2. ( J ) Model for the regulation of ATR/Chk1 activation in Tr2 DT. We propose that H 2 O 2 can induce ATR-dependent phosphorylation and activation of Chk1 in the absence of detectable DNA damage, leading to G2 arrest in Tr2 tracheoblasts. Scale bars = 10 µm.

    Article Snippet: The following antisera were used for immunohistochemical analysis: chicken anti-GFP (Aves, 1:500, RRID: AB_10000240 ), rabbit anti-phospho Chk1 (CST, 1:200, RRID: AB_331212 ), rabbit anti-pH3 (Millipore, 1:500, RRID: AB_310177 ), mouse anti-8-hydroxy-2′-deoxyguanosine (Abcam, 1:200, RRID: AB_867461 ), and Alexa 488/568-conjugated donkey anti-chicken/rabbit/mouse secondary antibodies (Invitrogen, 1:200).

    Techniques: Ex Vivo, Immunostaining, Expressing, Activation Assay

    Journal: eLife

    Article Title: Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts

    doi: 10.7554/eLife.68636

    Figure Lengend Snippet:

    Article Snippet: The following antisera were used for immunohistochemical analysis: chicken anti-GFP (Aves, 1:500, RRID: AB_10000240 ), rabbit anti-phospho Chk1 (CST, 1:200, RRID: AB_331212 ), rabbit anti-pH3 (Millipore, 1:500, RRID: AB_310177 ), mouse anti-8-hydroxy-2′-deoxyguanosine (Abcam, 1:200, RRID: AB_867461 ), and Alexa 488/568-conjugated donkey anti-chicken/rabbit/mouse secondary antibodies (Invitrogen, 1:200).

    Techniques: Amplification

    Journal: eLife

    Article Title: Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts

    doi: 10.7554/eLife.68636

    Figure Lengend Snippet:

    Article Snippet: The following antisera were used for immunohistochemical analysis: chicken anti-GFP (Aves, 1:500, RRID: AB_10000240 ), rabbit anti-phospho Chk1 (CST, 1:200, RRID: AB_331212 ), rabbit anti-pH3 (Millipore, 1:500, RRID: AB_310177 ), mouse anti-8-hydroxy-2′-deoxyguanosine (Abcam, 1:200, RRID: AB_867461 ), and Alexa 488/568-conjugated donkey anti-chicken/rabbit/mouse secondary antibodies (Invitrogen, 1:200).

    Techniques:

    Journal: eLife

    Article Title: Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts

    doi: 10.7554/eLife.68636

    Figure Lengend Snippet:

    Article Snippet: The following antisera were used for immunohistochemical analysis: chicken anti-GFP (Aves, 1:500, RRID: AB_10000240 ), rabbit anti-phospho Chk1 (CST, 1:200, RRID: AB_331212 ), rabbit anti-pH3 (Millipore, 1:500, RRID: AB_310177 ), mouse anti-8-hydroxy-2′-deoxyguanosine (Abcam, 1:200, RRID: AB_867461 ), and Alexa 488/568-conjugated donkey anti-chicken/rabbit/mouse secondary antibodies (Invitrogen, 1:200).

    Techniques: