Immunofluorescence staining of cell proteins is essential to understanding biomolecular interactions within three-dimensional (3D) hydrogel cell cultures. However, the scaffold material limits passive diffusion of antibodies through thick 3D matrices, prolonging staining and washing steps and resulting in processing times that can last for several days. Microwave irradiation has previously been shown to enhance penetration of fixatives in a variety of soft tissues by increasing the rate of diffusion through the sample, yet it is unknown if microwave irradiation can improve immunofluorescence staining of cells in 3D hydrogel cultures. Here, we demonstrate a microwave-assisted immunostaining technique that rapidly labels cells within spheroid structures embedded within thick intact hydrogels. These results show that collagen-embedded breast epithelial spheroids can be efficiently labeled with primary antibodies in less than 3.5 h. We show significantly enhanced staining and greater depth penetration with microwave-assisted staining compared to conventional benchtop staining methods. We demonstrate staining of collagen-embedded breast cancer spheroids with complete staining achieved in less than 2.5 h via the microwave, which outperforms conventional staining techniques. Moreover, we demonstrate enhanced staining of spheroids embedded in basement membrane-derived Matrigel matrices with the microwave method compared to benchtop techniques. Finally, we directly compare 2-h microwave-assisted staining to conventional 15-h longform benchtop staining and show that microwave staining increases depth penetration and intensity of stains compared to the longform staining. This work develops a microwave-assisted staining protocol that provides a rapid and reproducible method to label a variety of cell types within various 3D hydrogel cell culture systems.
© 2024 Author(s).

Lung cancer continues to be the leading cause of cancer-related deaths globally. Unraveling the regulators behind lung cancer growth and its metastatic spread, along with understanding the underlying mechanisms, is crucial for developing novel and effective therapeutic strategies. While much research has focused on identifying potential oncogenes or tumor suppressors, the roles of certain genes can vary depending on the context and may even exhibit contradictory effects. In this study, we demonstrate that acyl-CoA binding domain containing 3 (ACBD3), a Golgi resident protein, promotes primary lung cancer growth by recruiting phosphatidylinositol (PI)-4-kinase IIIβ (PI4KB) to the Golgi, thereby enhancing oncogenic secretion in chromosome 1q-amplified lung cancer cells. Conversely, in chromosome 1q-diploid lung cancer cells, ACBD3 acts as a suppressor of lung cancer metastasis by inhibiting the NOTCH signaling pathway and reducing cancer cell motility. This highlights the intricacy of cancer progression and cautions against simplistic approaches targeting individual oncogenes for cancer therapy.
© 2025. The Author(s).

The purpose of this study was to examine the nature of the underlying molecular mechanisms of cell death in human keratocytes treated with nigericin, a known pyroptosis inducer. Human keratocytes were exposed to nigericin, and cell death was assessed through morphological analysis and detection of related molecular markers. Proteomic profiling was performed to identify cell death-related proteins, with key findings validated by western blot. Additionally, organelle disruptions were examined using immunostaining techniques. Pyroptosis-like cell death was observed morphologically in cultured keratocytes. Moreover, an elevated release of IL-1beta was detected, accompanied by a significant loss of mitochondrial membrane potential. However, nigericin treatment induced a form of non-inflammatory cell death characterized by extensive vacuolation, resembling paraptosis. This was accompanied by the absence of caspase-3 activation and endoplasmic reticulum (ER) stress markers, along with increased accumulation of the autophagic marker LC3-II. Proteomic analysis revealed the absence of key components of the canonical pyroptosis pathway, including proteins involved in inflammasome assembly and the gasdermin (GSDM) family. These results were further confirmed by western blot. Significant alterations were also observed in the Golgi apparatus, mitochondria, and lysosomes following nigericin treatment. These findings suggest that nigericin triggers a paraptosis-like cell death in human keratocytes, rather than pyroptosis, as keratocytes lack the canonical executors of pyroptosis. This highlights an alternative mechanism of cell death in the cornea, warranting further exploration to understand its role and potential therapeutic implications.
© 2025 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.

Cancer cells exhibit heightened secretory states that drive tumor progression. Here, we identified a chromosome 3q amplicon that serves as a platform for secretory regulation in cancer. The 3q amplicon encodes multiple Golgi-resident proteins, including the scaffold Golgi integral membrane protein 4 (GOLIM4) and the ion channel ATPase secretory pathway Ca2+ transporting 1 (ATP2C1). We show that GOLIM4 recruited ATP2C1 and Golgi phosphoprotein 3 (GOLPH3) to coordinate Ca2+-dependent cargo loading, Golgi membrane bending, and vesicle scission. GOLIM4 depletion disrupted the protein complex, resulting in a secretory blockade that inhibited the progression of 3q-amplified malignancies. In addition to its role as a scaffold, GOLIM4 maintained intracellular manganese (Mn) homeostasis by binding excess Mn in the Golgi lumen, which initiated the routing of Mn-bound GOLIM4 to lysosomes for degradation. We show that Mn treatment inhibited the progression of multiple types of 3q-amplified malignancies by degrading GOLIM4, resulting in a secretory blockade that interrupted prosurvival autocrine loops and attenuated prometastatic processes in the tumor microenvironment. As it potentially underlies the selective activity of Mn against 3q-amplified malignancies, ATP2C1 coamplification increased Mn influx into the Golgi lumen, resulting in a more rapid degradation of GOLIM4. These findings show that functional cooperativity between coamplified genes underlies heightened secretion and a targetable secretory addiction in 3q-amplified malignancies.

The epicardium provides epicardial-derived cells and molecular signals to support cardiac development and regeneration. Zebrafish and mouse studies have shown that ccm2, a cerebral cavernous malformation disease gene, is essential for cardiac development. Endocardial cell-specific deletion of Ccm2 in mice has previously established that Ccm2 is essential for maintenance of the cardiac jelly for cardiac development during early gestation. The current study aimed to explore the function of Ccm2 in epicardial cells for heart development and regeneration. Through genetic deletion of Ccm2 in epicardial cells, our in vivo and ex vivo experiments revealed that Ccm2 is required by epicardial cells to support heart development. Ccm2 regulates epicardial cell adhesion, cell polarity, cell spreading, and migration. Importantly, the loss of Ccm2 in epicardial cells delays cardiac function recovery and aggravates cardiac fibrosis following myocardial infarction. Molecularly, Ccm2 targets the production of cytoskeletal and matrix proteins to maintain epicardial cell function and behaviors. Epicardial Ccm2 plays a critical role in heart development and regeneration via its regulation of cytoskeleton reorganization.
© 2024 The Authors.