Evaluation of Reference Gene Stability for Investigations of Intracellular Signalling in Human Cancer and Non-Malignant Mesenchymal Stromal Cells.

Background: Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful tool for analysing target gene expression in biological samples. To achieve reliable results by RT-qPCR, the most stable reference genes must be selected for proper data normalisation, particularly when comparing cells of different types. We aimed to choose the least variable candidate reference genes among eight housekeeping genes tested within a set of human cancer cell lines (HeLa, MCF-7, SK-UT-1B, A549, A431, SK-BR-3), as well as four lines of normal, non-malignant mesenchymal stromal cells (MSCs) of different origins.

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.

Positioning of endoplasmic reticulum exit sites around the Golgi depends on BicaudalD2 and Rab6 activity.

The endoplasmic reticulum (ER) is involved in biogenesis, modification and transport of secreted and membrane proteins. The ER membranes are spread throughout the cell cytoplasm as well as the export domains known as ER exit sites (ERES). A subpopulation of ERES is centrally localized proximal to the Golgi apparatus.

Functional cycle of EEA1-positive early endosome: Direct evidence for pre-existing compartment of degradative pathway.

Early endosomes, regarded as the main sorting station on endocytic pathway, are characterized by high frequency of homotypic fusions mediated by tethering protein EEA1. Despite intensive investigations, biogenesis of endosomes, boundaries between early and late endosomes, and process of cargo transition though them remain obscure. Here, using EGF/EGFR endocytosis as a model and confocal microscopy of fixed and live cells, we provide evidence favoring EEA1-vesicles being pre-existed vesicular compartment, that maintains its resident proteins' level and is sensitive to biosynthetic, but not endocytic pathway disturbance.

Paracrine senescence of human endometrial mesenchymal stem cells: a role for the insulin-like growth factor binding protein 3.

Stress-induced premature cell senescence is well recognized to be accompanied by emerging the senescence-associated secretory phenotype (SASP). Secreted SASP factors can promote the senescence of normal neighboring cells through autocrine/paracrine pathways and regulate the senescence response, as well. Regarding human endometrium-derived mesenchymal stem cells (MESCs), the SASP regulation mechanisms as well as paracrine activity of senescent cells have not been studied yet.

Quantitative analysis of the heterogeneous population of endocytic vesicles.

The quantitative characterization of endocytic vesicles in images acquired with microscope is critically important for deciphering of endocytosis mechanisms. Image segmentation is the most important step of quantitative image analysis. In spite of availability of many segmentation methods, the accurate segmentation is challenging when the images are heterogeneous with respect to object shapes and signal intensities what is typical for images of endocytic vesicles.

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.