Hippo overexpression decrease of lifespan and promotion of lipolysis in Drosophila. (A) Survivorship analysis of wild‐type (Dcg/+), Hippo‐overexpressing (Dcg > Hpo), and Hippo RNAi (Dcg > Hpo‐RNAi) D. melanogaster lines from newly eclosed male and female flies. N = 80. (B) Levels of TAG, DAG, and FFA species measured by TAG assay and HPLC‐MS/MS analyses of third instar larval homogenates isolated from Dcg/+ and Dcg > Hpo lines. Error bars represent SD of three independent experiments. N = 3; **p < 0.01; NS, not significant from Student's t‐test. (C, D) Level of DAG (C) and FFA (D) species measured by HPLC‐MS/MS of larval homogenates isolated from Dcg/+ and Dcg > Hpo lines. Error bars represent the SD of three independent experiments. N = 3; ***p < 0.001; **p < 0.01; *p < 0.05; NS, not significant from Student's t‐test. (E) Staining of fat bodies of third instar larvae from Dcg/+ and Dcg > Hpo lines using nile red (red) and DAPI labeling of nuclei (blue). Quantification of lipid droplet (LD) diameter in Dcg/+ and Dcg > Hpo animals. Each point represents a single LD. Error bars represent SD. N = 3, ***p < 0.001 from Student's t‐test. Scale bar, 50 μm. (F) Staining of fat bodies of third instar larvae from Dcg/+ and Dcg > Hpo animals using anti‐Pxn antibody as a marker of infiltrating immune cells. Scale bar, 50 μm. (G) Staining of fat bodies of third instar larvae from Dcg/+ and Dcg > Hpo animals using anti‐gamma‐H2AX antibody as a marker of senescence. Scale bar, 50 μm. (H) mRNA levels of dap and p53 from RT‐qPCR data of Dcg > Hpo. Error bars represent the SD of three independent experiments. N = 3, **p < 0.01, *p < 0.05 from Student's t‐test.