Fig. 5: Increase in ROS and loss of Lamin leads to collective puparium-generated EC death.

a Differentially expressed genes (p < 0.05, 1094 genes) between 4 d and 18 d UT midgut are classified based on GO terms for biological process and KEGG pathway. The microbiome defense and immune response categories are shown in red. P values was calculated by Fisher’s exact test. b Expression heatmap analysis is performed chronologically from 4 d to 18 d for the ROS metabolic and microbiome defense pathways of UT transcriptomes. c The schematic shows the method for monitoring ROS levels in puparium-generated ECs. ECs are transiently labeled with H2B-RFP prior to eclosion. The newly generated ECs in the adult are RFP- polyploid cells (blue nuclear ECs). d Representative images of gstD1-GFP staining in puparium-generated ECs (RFP⁺, white arrowhead) and newly generated ECs (RFP^-) in UT and EI midgut during aging. Note in the EI 30 d midgut, the boundary between gstD1-GFP intensity is the boundary between RFP⁺ and RFP^- ECs (the yellow dash line). e Statistics of gstD1-GFP intensity in RFP⁺ ECs or RFP^- ECs in 30 d EI midgut. n, numbers of ECs. f Statistics of the number of PH3⁺ cells in Myo1A^ts driving attp2 (control) and CncC RNAi midguts. n, number of midguts. n = 19 (attp2), 18 (CncC RNAi) midguts. g Representative images of Lamin staining in puparium-generated ECs (RFP⁺, white arrowhead) and newly generated ECs (RFP^-, yellow arrowhead) in the UT and EI midgut during aging. Close-up inset a’ shows strong Lamin staining in 4 d young RFP⁺ ECs, inset b’ shows Lamin staining is absent in RFP⁺ ECs but is restored in RFP^- ECs in 30 d EI midgut. h Statistics of Lamin intensity in RFP⁺ ECs during aging. n, numbers of ECs. i Statistics of Lamin intensity in RFP⁺ or RFP^- ECs in 30 d EI midguts. n, numbers of ECs. j Statistics of the number of PH3⁺ cells in Myo1A^ts driving attp2 (control) and Lamin RNAi midguts. n = 16 (attp2), 16 (Lamin RNAi) midguts. k A schematic summary of ROS and Lamin dynamics during EC aging and replacement. Data are mean ± SEM. Significance was determined using two-tailed unpaired t test. Scale bars, 10 μm. Source data are provided as a Source Data file.