Loss of Caspase-3 promotes cell death and induces autophagy in AE9a-expressing fetal liver cells. (A-B) Flow cytometric profiles (A) and statistical analysis of cell death (Annexin V⁻7AAD⁺) in MiGR1/AE9a-expressing fetal liver cells using Annexin V/7AAD assays. (C) Western blot of mTOR-autophagy signaling pathway proteins in MiGR1/AE9a-expressing fetal liver cell lysates. Compared with the AE9a-WT group, the protein levels of phosph-p70S6K and phosph-4EBP1 are lower compared with the AE9a-Caspase-3^−/− group. (D) Immunofluorescent staining of LC3 (red) in AE-expressing fetal liver cells and quantification of percentage of LC3⁺ cells. More LC3 punctate can be observed in the AE-Caspase-3^−/− group. (E) EM pictures of MiGR1/AE9a-expressing fetal liver cells and quantification of autophagic vesicles per cell. More autophagosomes and autolysosomes can be observed in the AE9a-Caspase-3^−/− group (scale bar, 10 μm). Red triangles represent autophagic vesicles.