Figure 10A. Quantification of three categories of midguts treated with the pH indicator Bromophenol Blue in Drosophila of indicated genotypes. The numbers of quantified guts from left to right are 90, 90, 90, 90, and 90. Error bars show the SD of three independent experiments. Data information: Error bars represent SDs. p-values for lifespan curves (C) were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. B. Quantification of excretion numbers of Drosophila with indicated genotypes. Excretions are quantified in 30 fields for each group of 12 Drosophila. Tests were repeated as three independent experiments. Data information: Error bars represent SDs. p-values for lifespan curves (C) were calculated by the log-rank test. The statistical tests used in other panels were 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. Survival (percentage) of female Drosophila of indicated genotypes. The numbers of quantified Drosophila: 100 (UAS-lacZ), 100 (Las RNAi), 100 (Las RNAi + UAS-Rab5-CA) and 100 (Las RNAi + UAS-Rab7-CA). Three independent experiments were conducted Data information: Error bars represent SDs. p-values for lifespan curves (C) were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. D. Quantification of three categories of midguts treated with the pH indicator Bromophenol Blue of Drosophila with indicated genotypes. The numbers of quantified guts from left to right are 90, 90, 90, and 90. Error bars show the SD of three independent experiments. Data information: Error bars represent SDs. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. E. Quantification of excretion numbers of Drosophila of indicated genotypes. Excretions are quantified in 30 fields for each group of 12 Drosophila. Tests were repeated as three independent experiments. Data information: Error bars represent SDs. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. F-I. Immunofluorescence images of dpERK staining of midgut sections of the R4 region of 40-day Drosophila (F), 40-day Drosophila with ALA administration (G), 14-day Drosophila carrying esgts-GAL4-driven UAS-lacZ (H), and Drosophila carrying esgts-GAL4-driven Las RNAi (I). dpERK (red). J. Quantification of the fluorescence intensity of dpERK in experiments (F-I). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 11, 12, 12, and 13. K Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm Error bars represent SDs. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. K-M. Representative images of midgut sections from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (K, control), Las RNAi (L), or Las RNAi and dominant negative form of EGFR (EGFR-DN, M). GFP (green) and Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm N. Quantification of esg-GFP+ cells, Dl+ cells, and pH3+ cells in experiments (K-M). n is indicated. The numbers of quantified guts from left to right are 16, 18, 19, 16, 18, 19, 16, 18, and 19. Data information: Error bars represent SDs. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. O. Quantification of luciferase activity of midguts of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA, Las RNAi, or Las RNAi and EGFR-DN. Error bars show the SD of six independent experiments. Data information: Error bars represent SDs. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05. P. Schematic model of the mechanism. In young flies highly expressed LAS synthesizes sufficient ALA in ISCs. ALA promotes endosome formation and maturation in ISCs, which in turn maintains the proper activation of autophagy and prevents the over activation of EGFR signaling in ISCs. As a secondary effect, ALA also prevents ISC overproliferation by scavenging excessive ROS. When flies get old, the expression of LAS dramatically reduces, which causes ALA deficiency and endosome reduction in ISCs. Ultimately, this leads to the hyperproliferation of ISCs with age.