(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 levels in arrested tracheoblasts. High levels of Chk1 expression are necessary for G2 arrest and Chk1 overexpression can rescue defects in Wnt signaling (Kizhedathu et al., 2020). (B, C) Effect of superoxide dismutase 1 (SOD1) overexpression and Dual oxidase (Duox) knockdown on expression of Wnts and Wnt-target genes. Quantitative PCR analysis of levels of Wg, Wnt5, Wnt6, Wnt10, Fz3, Chk1, and ATR mRNA in micro-dissected Tr2 DT fragments at L2. Graph shows fold change in Wg, Wnt5, Wnt6, Wnt10, Fz3, Chk1, and ATR mRNA levels in Tr2 dorsal trunk (DT) fragments expressing (B) btl-SOD1 (btl-GAL4/UAS-SOD1) and (C) btl-DuoxRNAi (btl-GAL4/+; UAS-DuoxRNAi (32903)/+). Fold change has been represented with respect to wild type (btl-Gal4, shown by dashed red line, n = 3 experiments, n ≥ 15 Tr2 DT fragments/stage/experiment, mean ± standard deviation). (D) Effect of overexpression of a phosphomimic variant of Chk1 in btl-DuoxRNAi larvae at 16–24 hr L3. Graph shows numbers of Tr2 tracheoblasts in wild type (btl-Gal4), btl-DuoxRNAi (btl-GAL4/+; UAS-DuoxRNAi (32903)/+), btl-DuoxRNAi, Chk1 (btl-GAL4/+; UAS-DuoxRNAi (32903)/ UAS-Chk1), btl-DuoxRNAi, ATR (btl-GAL4/+; UAS-DuoxRNAi (32903)/ UAS-ATR) and btl-DuoxRNAi, Chk1S373D (btl-GAL4/+; UAS-DuoxRNAi (32903)/ UAS-Chk1S373D) larvae at 16–24 hr L3 (n ≥ 7 tracheae per condition per timepoint). Student’s t-test: *p<0.00001.