Figure 1A. A model illustrating the Drosophila "esg>luciferase" reporter system of chemical administration. The chemicals were fed to Drosophila with esg-GAL4-driven luciferase expression on the 26th day after eclosion. After administration for 14 days, Drosophila were dissected, and the activity of luciferase in their midguts was measured. B. Quantification of the luciferase activity of flies with indicated ages and ALA administration. Error bars show the standard deviation (SD) of six independent experiments. Data information: Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. C-F. Immunofluorescence images of esg-GFP and Delta (Dl) staining with the midgut section from the R4 region of 14-day Drosophila (C), 26-day Drosophila (D), 40-day Drosophila (E), and 40-day Drosophila in response to 0.5 mM ALA administration, which started at day 26 after eclosion (F). esg-GFP (green) identifies ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. G. Quantification of the number of esg-GFP+ cells and Dl+ cells in experiments (C-F). n is indicated. The numbers of quantified guts from left to right are 15, 17, 16, 16, 15, 17, 16, and 16. Data information: Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. H. Quantification of the number of pH3+ cells in experiments (C-F). n is indicated. Data information: Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. I. Relative mRNA fold change of Las in sorted esg-GFP+ cells of wild-type (esg-GFP/CyO) Drosophila during aging. The Las expressions of Drosophila with different ages are plotted relative to 14-day Drosophila, which was set to 1. Error bars indicate the SD of three independent experiments. Data information: Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. J. Western blotting results of midguts indicate a decrease of LAS in the midguts of aged Drosophila. Loading controls, β-tubulin. K. Quantification of LAS band intensity as seen in experiments (J). Error bars indicate the SD of three independent experiments. L Data information: Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. L. Quantification of the content of ALA in midguts of 14-day, 26-day, and 40-day flies using LC-ESI-MS/MS. Error bars indicate the SD of three independent experiments. Data information: Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. M-O. LC-ESI-MS/MS chromatograms of ALA in midguts of 14-day flies(M), 26-day flies (N), and 40-day flies (O). Arrows indicate the peak of ALA. The area of ALA peak indicates the content of ALA. cps: counts per second. MRM: Multiple Reaction Ronitoring. When the estimated peak of ALA is undetected in the chromatogram, the content of ALA is considered as 0.