Duox-generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in Drosophila tracheoblasts
- Regulation of Cell Fate, Institute for Stem Cell Science and Regenerative Medicine (inStem), India
- National Centre for Biological Sciences, Tata Institute of Fundamental Research, India
- Trans Disciplinary University, India
Figures
Figure 1 with 2 supplements
High levels of reactive oxygen species (ROS) are required for checkpoint kinase 1 (Chk1) activation and G2 arrest in tracheoblasts.
(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 …
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Figure 1—source data 1
Cell Frequencies in SOD1 overexpressing animals.
- https://cdn.elifesciences.org/articles/68636/elife-68636-fig1-data1-v3.xlsx
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Figure 1—source data 2
Mitotic indices in SOD1 overexpressing animals.
- https://cdn.elifesciences.org/articles/68636/elife-68636-fig1-data2-v3.xlsx
Figure 1—figure supplement 1
Quantification of reactive oxygen species (ROS) levels in Tr2 tracheoblasts.
(A–F) Quantification of fluorescence intensities of redox-sensitive fluorescent dyes 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE) in Tr2 tracheoblasts. (A) …
Figure 1—figure supplement 2
Quantification of phosphorylated checkpoint kinase 1 (pChk1) levels in Tr2 tracheoblasts.
(A–C) Quantification of fluorescence intensities after pChk1 immunostaining and tyramide-based amplification. (A) Quantification of fluorescence intensities after pChk1 immunostaining in Tr2 …
Figure 2
High reactive oxygen species (ROS) in tracheoblasts is dependent on Duox expression.
(A) Quantitative PCR analysis of Duox mRNA levels in micro-dissected Tr2 dorsal trunk (DT) fragments at different stages. Graph shows fold change in Duox mRNA in Tr2 DT fragments from wild type (btl-…
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Figure 2—source data 1
Cell frequencies in Duox RNAi expressing animals.
- https://cdn.elifesciences.org/articles/68636/elife-68636-fig2-data1-v3.xlsx
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Figure 2—source data 2
Mitotic indices in Duox RNAi expressing animals.
- https://cdn.elifesciences.org/articles/68636/elife-68636-fig2-data2-v3.xlsx
Figure 3
Reactive oxygen species (ROS) dependence identifies a novel pathway for the regulation of ataxia telangiectasia mutated-related kinase/checkpoint kinase 1 (ATR/Chk1) in tracheoblasts.
(A) Model for G2 arrest mechanism in Tr2 tracheoblasts based on previous studies. Earlier work has shown that four Wnt ligands (Wg, Wnt5, Wnt6, Wnt10) act synergistically to upregulate Chk1 mRNA …
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Figure 3—source data 1
Cell frequencies in Chk1S373D expressing animals.
- https://cdn.elifesciences.org/articles/68636/elife-68636-fig3-data1-v3.xlsx
Figure 4 with 2 supplements
ATRIP, TOPBP1, and claspin are not required for reactive oxygen species (ROS)-mediated checkpoint kinase 1 (Chk1) activation in tracheoblasts.
(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 …
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Figure 4—source data 1
Cell frequencies in ATR RNAi, ATRIP RNAi, TOPBP1 RNAi and Claspin RNAi expressing animals.
- https://cdn.elifesciences.org/articles/68636/elife-68636-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
ATRIP, TOPBP1, and claspin are required for DNA damage-dependent activation ofATR/Chk1.
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) …
Figure 4—figure supplement 2
Loss of ATRIP, TOPBP1, and claspin does not affect cell numbers at WL3.
Effect of reduction of ATRIP, TOPBP1, or Claspin on cell numbers in Tr2 dorsal trunk (DT) at WL3. Graph shows cell numbers at WL3 in control (btl-GAL4), btl-ATRIPRNAi (btl-GAL4/UAS-ATRIPRNAi), btl-TO…
Figure 5 with 1 supplement
Incubation with H2O2 can restore phosphorylated checkpoint kinase 1 (pChk1) levels in Dual oxidase (Duox)-deficient tracheoblasts.
(A–E) Kinetics of Chk1 phosphorylation upon exposure to H2O2 ex vivo. (A) Regimen for H2O2 treatment and analysis of pChk1 in Tr2 dorsal trunk (DT) in L2. pChk1 immunostaining (red) in Tr2 DT in (B) …
Figure 5—figure supplement 1
Exposure to γ-radiation can restore phosphorylated checkpoint kinase 1 (pChk1) levels in dual oxidase (Duox)-deficient tracheoblasts.
(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-DuoxRNAi …
Tables
Key resources table
| Reagent type(species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Drosophila melanogaster) | btl-GAL4 | Shiga et al., 1996 | FLYB: FBtp0001208 | This line was a gift from Dr. Shigeo Hayashi |
| Genetic reagent (D. melanogaster) | UAS-Chk1RNAi | VDRC | 110076 | |
| Genetic reagent (D. melanogaster) | UAS-Chk1S373D | This study | Please see Materials and methods for a detailed description. (Can be obtained through NCBS Fly Facility: https://bangalorefly.ncbs.res.in/) | |
| Genetic reagent (D. melanogaster) | RPA-70GFP | Blythe and Wieschaus, 2015 | This line was a gift from Dr. Eric F Wieschaus | |
| Genetic reagent (D. melanogaster) | UAS-DuoxRNAi | BDSC | RRID:BDSC_33975 and RRID:BDSC_32903 | |
| Genetic reagent (D. melanogaster) | UAS-ATR | Bayer et al., 2018 | This line was a gift from Dr. Anja C Nagel | |
| Antibody | Phospho-Chk1 (Ser345) (rabbit monoclonal) antibody | CST | Cat #2348 (RRID:AB_331212) | (1:200) |
| Antibody | Anti-8-hydroxy-2’-deoxyguanosine antibody (mouse monoclonal) antibody | Abcam | Cat #ab48508 (RRID:AB_867461) | (1:200) |
| Commercial assay or kit | Tyramide signal amplification system | Thermo Fisher Scientific | Cat #B40912 |
