Prompt Description

Generate a technical roadmap that simplifies and organizes the experimental descriptions for clarity. The theme is the study of the downregulation mechanism of 14-3-3β in hypoxic stress of esophageal squamous cell carcinoma (ESCC) cells and its role in promoting ESCC progression. 1. Confirm the relationship between HIF1A and 14-3-3β. Related experiments include changes in HIF1A and 14-3-3β under different COCL2 concentrations and induction times, changes in 14-3-3β mRNA levels after induction, and changes in 14-3-3β after HIF1A knockdown and addition of the HIF1A inhibitor PX-478. 2. Phenotypic validation of 14-3-3β in ESCC cells, including MTT assays, qPCR, and colony formation assays. 3. Investigate the relationship between 14-3-3β and mitochondria, including changes in ROS detection, autophagy levels, mitochondrial membrane potential, and ATP levels. 4. Examine the relationship between 14-3-3β and ETFA, including co-IP, mitochondrial isolation, nucleocytoplasmic separation, and co-IP of mitochondrial and cytoplasmic proteins. 5. Elucidate the mechanism of action of 14-3-3β and ETFA and their impact on mitochondrial function, including changes in ETFA protein and mRNA levels after 14-3-3β knockdown, changes in cytoplasmic ETFA levels, mitochondrial oxygen consumption, changes in β-fatty acids, and changes in ferroptosis-related indicators. 6. In vivo experiments: CDX and inhibitor induction, tumor volume changes, IHC, and tissue microarray.