An annotated high-content fluorescence microscopy dataset with Hoechst 33342-stained nuclei and manually labelled outlines.

Automated detection of cell nuclei in fluorescence microscopy images is a key task in bioimage analysis. It is essential for most types of microscopy-based high-throughput drug and genomic screening and is often required in smaller scale experiments as well. To develop and evaluate algorithms and neural networks that perform instance or semantic segmentation for detecting nuclei, high quality annotated data is essential.

Methods to Detect Loss of Lysosomal Membrane Integrity.

Loss of lysosomal membrane integrity, often referred to as lysosomal membrane permeabilization (LMP), occurs in many instances of cell death either as an initiating or as an amplifying event. Currently, the best method for detecting LMP is the galectin puncta formation assay which can be used for a broad range of sample types, both fixed and live, is easy to perform, and highly sensitive. This method, which is similar to the widely used LC3 puncta formation assay for autophagy, is based on the translocation of galectins to damaged lysosomes resulting in a change from uniform to punctate staining pattern.

Discovery of Small Molecules That Induce Lysosomal Cell Death in Cancer Cell Lines Using an Image-Based Screening Platform.

The lysosomal cell death (LCD) pathway is a caspase 3-independent cell death pathway that has been suggested as a possible target for cancer therapy, making the development of sensitive and specific high-throughput (HT) assays to identify LCD inducers highly desirable. In this study, we report a two-step HT screening platform to reliably identify such molecules. First, using a robust HT primary screen based on propidium iodide uptake, we identified compounds that kill through nonapoptotic pathways.

Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay.

Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP.

Methods for the quantification of lysosomal membrane permeabilization: a hallmark of lysosomal cell death.

Lysosomal cell death is triggered by lysosomal membrane permeabilization (LMP) and subsequent release of lysosomal hydrolases from the lysosomal lumen into the cytosol. Once released into the cytosol, the lysosomal cathepsin proteases act as executioner proteases for the subsequent cell death-either autonomously without caspase activation or in concert with the classical apoptotic machinery. Lysosomal cell death usually remains functional in apoptosis-resistant cancer cells and thus holds great potential as a therapeutic strategy for circumventing apoptosis deficiency in cancers.

Lysosomal cell death at a glance.

Lysosomes serve as the cellular recycling centre and are filled with numerous hydrolases that can degrade most cellular macromolecules. Lysosomal membrane permeabilization and the consequent leakage of the lysosomal content into the cytosol leads to so-called "lysosomal cell death". This form of cell death is mainly carried out by the lysosomal cathepsin proteases and can have necrotic, apoptotic or apoptosis-like features depending on the extent of the leakage and the cellular context.

Identification of cytoskeleton-associated proteins essential for lysosomal stability and survival of human cancer cells.

Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death.

HAMLET binding to α-actinin facilitates tumor cell detachment.

Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents.

Changes in proteasome structure and function caused by HAMLET in tumor cells.

Background: Proteasomes control the level of endogenous unfolded proteins by degrading them in the proteolytic core. Insufficient degradation due to altered protein structure or proteasome inhibition may trigger cell death. This study examined the proteasome response to HAMLET, a partially unfolded protein-lipid complex, which is internalized by tumor cells and triggers cell death.

HAMLET (human alpha-lactalbumin made lethal to tumor cells) triggers autophagic tumor cell death.

HAMLET, a complex of partially unfolded alpha-lactalbumin and oleic acid, kills a wide range of tumor cells. Here we propose that HAMLET causes macroautophagy in tumor cells and that this contributes to their death. Cell death was accompanied by mitochondrial damage and a reduction in the level of active mTOR and HAMLET triggered extensive cytoplasmic vacuolization and the formation of double-membrane-enclosed vesicles typical of macroautophagy.

Can misfolded proteins be beneficial? The HAMLET case.

By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain.

Recurrent and multiple bladder tumors show conserved expression profiles.

Background: Urothelial carcinomas originate from the epithelial cells of the inner lining of the bladder and may appear as single or as multiple synchronous tumors. Patients with urothelial carcinomas frequently show recurrences after treatment making follow-up necessary. The leading hypothesis explaining the origin of meta- and synchronous tumors assumes a monoclonal origin.

Apoptosis and tumor cell death in response to HAMLET (human alpha-lactalbumin made lethal to tumor cells).

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a molecular complex derived from human milk that kills tumor cells by a process resembling programmed cell death. The complex consists of partially unfolded alpha-lactalbumin and oleic acid, and both the protein and the fatty acid are required for cell death. HAMLET has broad antitumor activity in vitro, and its therapeutic effect has been confirmed in vivo in a human glioblastoma rat xenograft model, in patients with skin papillomas and in patients with bladder cancer.