The Dual Luminescence Lifetime pH/Oxygen Sensor: Evaluation of Applicability for Intravital Analysis of 2D- and 3D-Cultivated Human Endometrial Mesenchymal Stromal Cells.

The oxygenation of cells and tissues and acidification of the cellular endolysosomal system are among the major factors that ensure normal functioning of an organism and are violated in various pathologies. Recording of these parameters and their changes under various conditions is an important task for both basic research and clinical applications. In the present work, we utilized internalizable dual pH/O lifetime sensor (Ir-HSA-FITC) based on the covalent conjugation of human serum albumin (HSA) with fluorescein isothiocyanate (FITC) as pH sensor and an orthometalated iridium complex as O sensor.

EGF, TGF- and Amphiregulin Differently Regulate Endometrium-Derived Mesenchymal Stromal/Stem Cells.

The prototypical receptor tyrosine kinase epidermal growth factor receptor (EGFR) is regulated by a set of its ligands, which determines the specificity of signaling and intracellular fate of the receptor. The EGFR signaling system is well characterized in immortalized cell lines such as HeLa derived from tumor tissues, but much less is known about EGFR function in untransformed multipotent stromal/stem cells (MSCs). We compared the effect of epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and amphiregulin (AREG) on physiological responses in endometrial MSCs (enMSC) and HeLa cells.

Machine Learning Assisted Classification of Cell Lines and Cell States on Quantitative Phase Images.

In this report, we present implementation and validation of machine-learning classifiers for distinguishing between cell types (HeLa, A549, 3T3 cell lines) and states (live, necrosis, apoptosis) based on the analysis of optical parameters derived from cell phase images. Validation of the developed classifier shows the accuracy for distinguishing between the three cell types of about 93% and between different cell states of the same cell line of about 89%. In the field test of the developed algorithm, we demonstrate successful evaluation of the temporal dynamics of relative amounts of live, apoptotic and necrotic cells after photodynamic treatment at different doses.

Biocompatible Ir(III) Complexes as Oxygen Sensors for Phosphorescence Lifetime Imaging.

Synthesis of biocompatible near infrared phosphorescent complexes and their application in bioimaging as triplet oxygen sensors in live systems are still challenging areas of organometallic chemistry. We have designed and synthetized four novel iridium [Ir(N^C)(N^N)] complexes (N^C-benzothienyl-phenanthridine based cyclometalated ligand; N^N-pyridin-phenanthroimidazol diimine chelate), decorated with oligo(ethylene glycol) groups to impart these emitters' solubility in aqueous media, biocompatibility, and to shield them from interaction with bio-environment. These substances were fully characterized using NMR spectroscopy and ESI mass-spectrometry.

EGF receptor lysosomal degradation is delayed in the cells stimulated with EGF-Quantum dot bioconjugate but earlier key events of endocytic degradative pathway are similar to that of native EGF.

Quantum dots (QDs) complexed to ligands recognizing surface receptors undergoing internalization are an attractive tool for live cell imaging of ligand-receptor complexes behavior and for specific tracking of the cells of interest. However, conjugation of quasi-multivalent large QD-particle to monovalent small growth factors like EGF that bound their tyrosine-kinase receptors may affect key endocytic events tightly bound to signaling. Here, by means of confocal microscopy we have addressed the key endocytic events of lysosomal degradative pathway stimulated by native EGF or EGF-QD bioconjugate.

Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells.

Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis.

Quantum dots implementation as a label for analysis of early stages of EGF receptor endocytosis: a comparative study on cultured cells.

EGF complexed to fluorescent photostable quantum dots by biotin-streptavidin system (bEGF-savQD) is attractive for both the basic research and therapeutic application such as targeted drug delivery in EGF-receptor (EGFR) expressing cancers. However, compared to native EGF, the large size of QD and its quasi-multivalency can have unpredictable effects on EGFR endocytosis changing the internalization portal and/or endosomal processing tightly bound to EGF signaling. We have found that bEGF-savQDs enter HeLa cells via the temperature-dependent clathrin-mediated EGF-receptor-specific pathway characteristic for native EGF.

Termination of tyrphostin AG1478 application results in different recovery of EGF receptor tyrosine residues 1045 and 1173 phosphorylation in A431 cells.

Tyrphostin AG1478 is known as a specific and reversible inhibitor of TK (tyrosine kinase) activity of the EGFR [EGF (epidermal growth factor) receptor]. It is attractive as an anticancer agent for cancers with elevated EGFR TK levels. However, post-application effects of AG1478 are not well studied.

Epidermal growth factor (EGF) receptor endocytosis is accompanied by reorganization of microtubule system in HeLa cells.

Translocation of endosomes along microtubules (MTs) from the cell periphery toward the juxtranuclear region proximal to MTOC is well established. During this translocation the radial MT system is believed to retain its organization. Here we demonstrate that epidermal growth factor receptor (EGFR) endocytosis in HeLa cells is accompanied by dramatic remodeling of the MT system.

Two different stages of epidermal growth factor (EGF) receptor endocytosis are sensitive to free ubiquitin depletion produced by proteasome inhibitor MG132.

Numerous studies implicate proteasomes in the regulation of EGF receptor (EGFR) endocytosis on the basis of the ability of inhibitors to decrease EGFR degradation, but the exact mechanisms remain obscure. We demonstrated that EGFR itself is not a direct target for proteasome, since it is delivered to lysosomes intact. Evidence is presented that the inhibitory effect of MG132 on EGF degradation is due mostly to free ubiquitin depletion resultant from the suppression of proteasomal functioning by MG132.