Development of Immunoelectron Microscopy for VASN Detection Using Multiple Models and Identification of VASN as a Key Regulator of Mitochondrial Homeostasis in Hepatocellular Carcinoma Cells.

Vasorin (VASN), a transmembrane glycoprotein, plays a pivotal role in hepatocellular carcinoma (HCC) progression by regulating cell proliferation and migration. However, its subcellular localization and organelle-specific functional mechanisms remain inadequately defined, mainly due to the limitations of ultrastructural resolution and quantitative analytical techniques. In this study, an optimized immunoelectron microscopy protocol (IEM-VASN) was developed, combining 4% paraformaldehyde-0.5% glutaraldehyde fixation and a 1:200 anti-VASN antibody dilution, achieving an optimal balance between structural preservation and antigenicity. This method enabled the precise detection of VASN depletion in Huh7-KD cells (p < 0.0001) and Vasn-/- mice (p < 0.0001). IEM-VASN revealed VASN's organelle-specific distribution, showing significant enrichment in mitochondria in both HCC cells and liver tissues (p < 0.001 and p < 0.0001). Furthermore, murine HCC tissues displayed elevated VASN levels (p < 0.001) alongside reduced mitochondrial counts (p < 0.01). In a chronic aflatoxin B1 (AFB1) exposure model, VASN upregulation correlated with ROS accumulation, mitochondrial membrane potential dissipation, and mitophagy induction. The IEM-VASN method demonstrated high precision and applicability across cellular, animal, and pathological models, providing a powerful tool for elucidating the functional roles of VASN in HCC. These findings establish a highly sensitive and specific IEM technique for characterizing VASN's ultrastructural distribution, highlighting its predominant mitochondrial localization and functional involvement in HCC progression and chemically-induced mitophagy, thus positioning VASN as a critical molecular mediator in liver cancer and mitochondrial homeostasis.