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Abstract Various intracellular degradation organelles, including autophagosomes, lysosomes, and endosomes, work in tandem to perform autophagy, which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. This paper aims to describe an approach to reproducibly identify and distinguish subcellular structures involved in macroautophagy. Methods are provided that help avoid common pitfalls. How to distinguish between lysosomes, lipid droplets, autolysosomes, autophagosomes, and inclusion bodies are also discussed. These methods use transmission electron microscopy (TEM), which is able to generate nanometer‐scale micrographs of cellular degradation components in a fixed sample. Serial block face‐scanning electron microscopy is also used to visualize the 3D morphology of degradation machinery using the Amira software. In addition to TEM and 3D reconstruction, other imaging techniques are discussed, such as immunofluorescence and immunogold labeling, which can be used to classify cellular organelles, reliably and accurately. Results show how these methods may be used to accurately quantify cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin‐related protein 1. 

Abstract Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high‐resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods are used to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, andDrosophilamuscles. The accuracy of assessment is validated in cells and tissue with deletion of genes involved in mitochondrial dynamics. 

The Melanoma Antigen Gene (MAGE) family of proteins is the largest family of cancer-testis antigens (CTAs) and shares a MAGE homology domain (MHD). MAGE proteins are divided into Type I and Type II MAGEs depending on their chromosomal location and expression patterns. Type I MAGEs are true CTAs. MAGEB2 is a Type I MAGE, belonging to the MAGEB subfamily, and unlike some MAGE proteins, has not been found to bind to and enhance E3 ligase activity. MAGEB2 has been discovered to be an RNA-binding protein that serves to protect spermatogonial cells in the testis from extraneous stressors. We have discovered that MAGEB2 is necessary and sufficient for the proliferation of cells and is expressed by the differential DNA methylation of its gene promoter. Furthermore, we identified JunD as the transcription factor that regulates MAGEB2 expression. When expressed, MAGEB2 suppresses transforming grown factor-β1 (TGFβ1) signaling by decreasing mRNA levels of Thrombospondin-1 (TSP-1). TSP-1 is an anti-angiogenic protein that activates TGFβ1. Restoring levels of TSP-1 or TGFβ1 results in the inability of MAGEB2 to drive proliferation, suggesting that MAGEB2-expressing tumors might be more susceptible to therapies that induce or activate TSP-1 or TGFβ1 signaling.