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. 2021 Oct 8;10:e68636. doi: 10.7554/eLife.68636

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© 2021, Kizhedathu et al

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Figure 5. — (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) untreated btl-Duox^RNAi (btl-GAL4/+; UAS-Duox^RNAi (32903)/+)-expressing tracheae and treated with 100 µM H₂O₂ 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 H₂O₂ ex vivo. pChk1 immunostaining (red) in Tr2 DT in btl-ATR^RNAi (btl-GAL4/UAS-ATR^RNAi) tracheae treated with 100 µM H₂O₂ 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 H₂O₂ 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 H₂O₂ for 2 min at L2. (J) Model for the regulation of ATR/Chk1 activation in Tr2 DT. We propose that H₂O₂ 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 5—figure supplement 1. — (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) untreated btl-Duox^RNAi (btl-GAL4/+; UAS-Duox^RNAi (32903)/+)-expressing tracheae and treated with 100 µM H₂O₂ 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 H₂O₂ ex vivo. pChk1 immunostaining (red) in Tr2 DT in btl-ATR^RNAi (btl-GAL4/UAS-ATR^RNAi) tracheae treated with 100 µM H₂O₂ 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 H₂O₂ 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 H₂O₂ for 2 min at L2. (J) Model for the regulation of ATR/Chk1 activation in Tr2 DT. We propose that H₂O₂ 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.