Methods in molecular biology最新文献

During the last three decades, technological advancements in high-throughput next-generation sequencing have resulted in an increased understanding of proteomic and genomic data, aptly termed proteogenomics. Efforts in developing such approaches have not only been limited but also focused on protein identification and subcellular localization. These approaches, however, have also been explored for their broad understanding of how genomics/transcriptomics data have yielded measures, for example, gene expression regulation/signal cascading and diseasome studies. In this review, we discuss methods and tools developed through sequence-centric integrative modeling of proteogenomic approaches.

Autophagy is a vital cellular process responsible for breaking down faulty cellular components and organelles, ultimately routed through lysosomes for degradation. This intricate mechanism involves the translocation of LC3, a cytoplasmic protein, onto the autophagosome membranes. As a result, it becomes feasible to discern cells engaged in autophagy by employing fluorescent markers designed for LC3 or other indicative autophagy markers. Although a variety of techniques such as immunofluorescence and western blotting serve as indispensable tools for assessing autophagy, the definitive confirmation comes from the visualization of autophagosomes using transmission electron microscopy. While numerous protocols for antibody staining can be found in scientific literature and on antibody suppliers' websites, these procedures often demand significant time and financial resources for setup. This chapter endeavors to provide a user-friendly and cost-effective guide for practitioners seeking proficiency in immunofluorescence staining and western blotting techniques.

A type of three-dimensional (3D) cell culture models which is simple and easy is hanging drop method. The hanging drop method emerges as a pivotal technique with diverse applications in cancer research and cell biology. This method facilitates the formation of multicellular spheroids, providing a unique environment for studying cell behavior dynamics. The hanging drop method's theoretical underpinning relies on gravity-enforced self-assembly, allowing for cost-effective, reproducible 3D cell cultures with controlled spheroid sizes. The advantages of this approach include its efficiency in producing cellular heterogeneity, particularly in non-adherent 3D cultures, and its ability to create hypoxic spheroids, making it a suitable model for studying cancer. Moreover, the hanging drop method has proven valuable in investigating various aspects such as tissue structure, signaling pathways, immune activation of cancer cells, and notably, cell proliferation. Researchers have utilized the hanging drop method to explore the dynamics of cell proliferation, studying the effects of mesenchymal stem cells (MSC) secretome on cancer cells. The method's application involves co-culturing different cell lines, assessing spheroid formations, and quantifying their sizes over time. These studies have unveiled intricate cell behavior dynamics, demonstrating how the MSC secretome influences cancer cell growth and viability within a three-dimensional co-culture paradigm.

The exquisite balance between cellular prosurvival and death pathways is extremely necessary for homeostasis. Different forms of programmed cell death have been widely studied and reported such as apoptosis, necroptosis, pyroptosis, and autophagy. Autophagy is a catabolic process important for normal cellular functioning. The main aim of this machinery is to degrade the misfolded or damaged proteins, unuseful organelles, and pathogens, which invade the cells, thereby maintaining cellular homeostasis and assuring the regular renewal of cell components. This prosurvival function of autophagy highlights its importance in many human diseases, as the disturbance of this tightly organized process ultimately causes detrimental effects. Interestingly, neurons are particularly susceptible to damage upon the presence of any alteration in the basal level of the autophagic activity; this could be due to their high metabolic demand, post-mitotic nature, and the contribution of autophagy in the different fundamental functions of neurons. Herein, we have reported the role of autophagy in different CNS disorders such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and epilepsy, besides the pharmacological agents targeting autophagy. Due to the significant contribution of autophagy in the pathogenesis of many diseases, it is crucial to develop effective methods to detect this dynamic process. In this chapter, we have summarized the most frequently employed techniques in studying and detecting autophagy including electron microscopy, fluorescence microscopy, Western blotting, intracellular protein degradation, and sequestration assay.

Non-small cell lung cancer (NSCLC) is among the most malignant tumors with high propensity for metastasis and is the leading cause of cancer-related death globally. Most patients present with regional and distant metastasis, associated with poor prognosis. Lipids may play an essential role in either activating or inhibiting detachment-induced apoptosis (anoikis), where the latter is a crucial mechanism to prevent metastasis, and it may have a cross-talk with autophagy. Autophagy has been shown to be induced in various human cancer metastasis, modulating tumor cell motility and invasion, cancer cell differentiation, resistance to anoikis, and epithelial to mesenchymal transition. Hence, it may play a crucial role in the transition of benign to malignant phenotypes, the core of metastasis initiation. Here, we provide a method we have established in our laboratory for detecting lipids in attached and detached non-small lung cancer cells and show how to analyze lipidomics data to find its correlation with autophagy-related pathways.

The role of shear stress in regulating aqueous humor (AH) outflow and intraocular pressure (IOP) in the trabecular meshwork (TM) and Schlemm's canal (SC) of the eye is an emerging field. Shear stress has been shown to activate mechanosensitive ion channels in TM cells and induce nitric oxide production in SC cells, which can affect outflow resistance and lower IOP. Live-cell imaging using fluorescent protein sensors has provided real-time data to investigate the physiological relationship between fluid flow and shear stress in the outflow pathway cells. The successful application of time-lapse live-cell imaging in primary cultured cells has led to the identification of key cellular and molecular mechanisms involved in regulating AH outflow and IOP, including the role of autophagy and primary cilia as mechanosensors. This chapter presents a detailed protocol for conducting time-lapse live-cell imaging under fluid flow conditions in the outflow pathway cells.

The nematode Caenorhabditis elegans, widely recognized as a model organism due to its ease of breeding and well-characterized genomes, boasts complete digestive, reproductive, and endocrine systems, as well as conserved signaling pathways shared with mammals. It has become an invaluable resource for metabolomics research, particularly in examining responses to chemical or environmental factors and toxicity assessments. In this article, we provide detailed, step-by-step protocols for cultivating C. elegans and conducting metabolomics analyses, specifically focusing on sample preparation for GC-MS analysis in response to toxic compound treatments. We highlight the critical choice of extraction solvent, introducing two representative systems for extracting metabolites from C. elegans.

Feedback regulation of squalene monooxygenase (SM), a rate-limiting enzyme of cholesterol synthesis, is an important component of cellular lipid homeostasis. This regulation is exerted in part by the cholesterol-induced degradation of the SM protein. Here, we describe the cell culture, sample preparation, and immunoblotting conditions that our laboratory routinely uses to study the cholesterol regulation of both endogenous and ectopic SM. We also provide a worked example of quantifying the cholesterol-induced degradation of SM using densitometry.

The mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient-sensing complex that integrates inputs from several pathways to promote cell growth and proliferation. mTORC1 localizes to many cellular compartments, including the nucleus, lysosomes, and plasma membrane. However, little is known about the spatial regulation of mTORC1 and the specific functions of mTORC1 at these locations. To address these questions, we previously developed a Förster resonance energy transfer (FRET)-based mTORC1 activity reporter (TORCAR) to visualize the dynamic changes in mTORC1 activity within live cells. Here, we describe a detailed protocol for using subcellularly targeted TORCAR constructs to investigate subcellular mTORC1 activities via live-cell fluorescence microscopy.