Identification of protein components of the transformation system in the cell line of immortalized human keratinocytes HaCaT exposed to surfactants.

Using the method of shotgun mass spectrometry, we have evaluated changes in the proteomic profile of HaCat cells in response to the treatment with sodium dodecyl sulfate (anionic surfactant) and Triton-X100 (non-ionic surfactant) in two concentrations (12.5 µg/ml and 25.0 µg/ml). The study revealed induction of orphan CYP2S1 (biotransformation phase I) in response to Triton-X100. We have identified proteins of II (glutathione-S-transferases, GSTs) and III (solute carrier proteins, SLCs) biotransformation phases, as well as antioxidant proteins (peroxiredoxins, PRDXs; catalase, CAT; thioredoxin, TXN). Thus, proteins of all three xenobiotic detoxification phases were detected. The presented results suggest a new prospect of using HaCaT keratinocytes as a model of human epidermis for studying the metabolism of drugs/toxicants in human skin in vitro.

System analysis of surface CD markers during the process of granulocytic differentiation.

Plasma membrane proteins with extracellular-exposed domains are responsible for transduction of extracellular signals into intracellular responses, and their accessibility to therapeutic molecules makes them attractive targets for drug development. In this work, using omics technologies and immunochemical methods, we have studied changes in the content of markers of clusters of differentiation (CD markers) of neutrophils (CD33, CD97, CD54, CD38, CD18, CD11b, CD44, and CD71) at the level of transcripts and proteins in NB4, HL-60 and K562 cell lines, induced by the treatment with all-trans-retinoic acid (ATRA). Transcriptomic analysis revealed the induction of CD38, CD54, CD11b, and CD18 markers as early as 3 h after the addition of the inducer in the ATRA-responsive cell lines HL-60 and NB4. After 24 h, a line-specific expression pattern of CD markers could be observed in all cell lines. Studies of changes in the content of CD antigens by means of flow cytometry and targeted mass spectrometry (MS) gave similar results. The proteomic profile of the surface markers (CD38, CD54, CD11b, and CD18), characteristic of the NB4 and HL-60 lines, reflects different molecular pathways for the implementation of ATRA-induced differentiation of leukemic cells into mature neutrophils.

Phenotypic Features of Mesenchymal Stem Cell Subpopulations Obtained under the Influence of Various Toll-Like Receptors Ligands.

The paper characterizes phenotypic features of subpopulations of human adipose tissue mesenchymal stem cells (MSC) resulting from exposure to Toll-like receptors ligands. MSC1 and MSC2 phenotypes were induced by exposure to LPS and polyinosinic-polycytidylic acid, respectively. Different exposures to Toll-like receptors ligands, short-term (3 h) and long-term (24 h), were studied. The cytokine profile of cells with the MSC2 phenotype differed depending on the duration of exposure to the inductor, while the cytokine profile of cells with the MSC1 phenotype remained stable. Morphometric features of mesenchymal stem cells with the MSC1 and MSC2 phenotypes, as well as differences in the IDO gene expression were identified. These differences can be used to distinguish between these cell types.

Activation of TLR4 of Mesenchymal Stem Cells Enhances the Regenerative Properties of Their Secretomes.

Mesenchymal stem cells (MSC), like macrophages, can be polarized in vitro. In particular, activation of type 4 Toll-like receptor in MSC leads to the appearance of the so-called "proinflammatory" MSC phenotype (MSC1). We showed that secretome (conditioned media) of MSC1 can affect the wound healing processes: promote healing and modulate exudative inflammation and subsequent fibroplastic processes in the damaged area. These effects of secretomes of polarized MSC were superior to those of intact MSC.

Protein dataset of immortalized keratinocyte HaCaT cells and normal human keratinocytes.

Learning of the molecular mechanisms of the pathological processes development in the normal human keratinocytes (NHK) are difficult. Immortalized keratinocytes HaCaT are often used as an analogue of NHK since they have a number of advantages over the latter - they do not require the presence of growth and differentiation factors in the medium, have unlimited potential for proliferation, demonstrate stable phenotype regardless of the number of passages [1]. Taking into account the properties and characteristics of the HaCaT line, these cells can be considered as a promising experimental model for research of various physiological processes occurring in human keratinocytes. However, to understand the limitations of such an experimental model, a detailed comparative characterization of HaCaT and NHK is required, which can be obtained by carrying out its proteomic analysis. In this article we present datasets obtained through the high-throughput shotgun proteomics analysis of normal human keratinocytes and immortalized HaCaT keratinocytes. As a protocol for proteomic profiling of cells, we used the approach of obtaining LC-MS / MS measurements followed by their processing with Progenesis LC-MS software (Nonlinear Dynamics Ltd.). The mzML files were deposited to the Mendeley Data.

[Comparative study of the human keratinocytes proteome of the HaCaT line: identification of proteins encoded by genes of 18 chromosomes under the influence of detergents]. / Sravnitel'noe issledovanie proteoma kletok HaCaT keratinotsitov cheloveka: identifikatsiia belkov, kodiruemykh genami 18 khromosomy pri vozdeistvii detergentov.

Using electrospray ionization tandem mass spectrometry, a comparative analysis of the HaCaT keratinocyte proteins encoded by the 18th chromosome was performed before and after exposure to sodium dodecyl sulfate (25 mg/ml) and to Triton X-100 (12.5 mg/ml) in a subtoxic dose for 48 hours. Proteins were identified using the SearchGUI platform (X!Tandem and MS-GF+ search engines). In total, 1284 proteins were found in immortalized human HaCaT keratinocytes and about 75% of them were identified by two or more peptides. Were identified, that 26 proteins were encoded by genes of chromosome 18. Among these proteins, 17 were common for control cells and HaCaT cells treated with SDS. Proteins MARE2 and CTIF were identified only in control keratinocytes. Seven identified proteins encoded by genes of chromosome 18 were found only in detergent-treated keratinocytes: LMAN1, NDUV2, SPB3, VPS4B, KDSR, ROCK1 and RHG28.

Proteome dataset of mouse macrophage cell line infected with tick-borne encephalitis virus.

We report the proteomic datasets on the mouse macrophage cell line PMJ2R infected with tick-borne encephalitis virus (TBEV) for two and six days. Data were acquired using shotgun ultra-high resolution mass spectrometry. Peptide identifications were done using the Mascot version 2.4 (Matrix Science), and quantification was performed by a label-free approach. Protein profiles of early (two days) and late (six days) stages of infection were compared between each other and the respective control samples. Protein profiles of infected and control samples differed in the number of identified proteins and their relative abundances. Proteins detected in the TBEV-infected cells were involved in various processes related to the infection, including defense response against the virus, regulation of viral process, negative regulation of viral genome replication, RNA binding, or innate immune response. Also, proteins specific for the early and late stages of infection were identified.

A Cell Model of Human Small Intestinal Wall Based on Genetically Modified Caco-2 Cells.

We propose a cell model of the human small intestinal wall based on genetically modified Caco-2 cells that allows visualization and quantitative assessment of activation of NF-κB factor and related intracellular pathway by using fluorescence microscopy. A dose-dependent increase in fluorescence intensity of the obtained cells in response to TNFα exposure in concentrations of 1-100 ng/ml was demonstrated. It was found that this parameter correlates with a decrease in the transepithelial resistance of the cell monolayer in response to TNFα and can be used to assess the toxic effects of substances on epithelial cells of the human small intestine.

[Quality control study of engineered skin tissue]. / Metod otsenki kachestva rekonstruirovannogo épidermisa.

OMERO service was used to annotate the cell line HaCaT microscope images by two independent expert groups. The images were obtained in the course of developing tissue-engineered epithelium which consisted of several layers of the keratinocytes. Evaluation of expert opinions was performed by calculation of specificity, sensitivity and accuracy. The best convergence of opinions (91%) was achieved for the confluence of the cell monolayers. Accuracy 70% was observed in determining the extent of cell differentiation after 10 days of incubation. The paper illustrates the usefulness of OMERO service for dynamic cross-validation of quality in the development and standardization of cell preparations.

[Proteome of the human HaCaT keratinocytes: Identification of the oxidative stress proteins after sodium dodecyl sulpfate exposur].

Oxidative stress is a universal response of the skin cell damage of various origins. Sodium dodecyl sulfate (SDS, sodium lauryl sulfate) is an anionic surfactant commonly used as an emulsifying detergent in household cleaners. Sodium dodecyl sulfate is the reference compound for testing toxicity on cellular skin models. The effect of sodium dodecyl sulfate in sub toxic dose 25 µg/mL during 48 h on the protein profile of human keratinocytes HaCaT was studied by tandem mass spectrometry with electrospray ionization. In total, 1064 proteins were found in immortalized human keratinocytes HaCaT, of which about 80% were identified by two or more peptides. The change of the 217 proteins content was revealed, among them 39 according to Gene Ontology are associated with oxidative stress. It has been found that sodium dodecyl sulfate leads to a decrease in the number of proteins/peptides containing carboxymethylated and/or carboxyethylated lysine. We concluded about the promising of the cells redox-balance analysis at testing chemicals in the doses, which do not lead to a decrease in their viability. Possible involvement of sodium dodecyl sulfate in the development of cutaneous neoplasia is discussed.

[Proteomic profiling of HaCaT keratinocytes exposed to skin damaging detergents]. / Proteomnoe profilirovanie keratinotsitov linii HaCaT pri vozdeistvii riada poverkhnostno-aktivnykh veshchestv, vyzyvaiushchikh povrezhdenie kozhi.

The effects of sodium dodecyl sulfate (25 mg/ml) and Triton X-100 (12.5 mg/ml and 25 mg/ml) on the HaCaT immortalized keratinocytes exposed to these surfactants for 48 h were studied. Using shotgun proteomics, a comparative analysis of the proteomic profiles of control and experimental cells after surfactants exposure was carried out. 260 common proteins were identified in control and experimental cells; 33 proteins were found in cells exposed to all three treatments, but not in control cells. These 33 proteins apparently reflect a nonspecific (universal) response of cells to toxic damage by the surfactants. These proteins are associated with activation of cell proliferation, changes in the functional activity of their ER and mitochondria, increased mRNA stability and activation of protein degradation processes in the cells. The possibility of using these proteins as a nonspecific parameter of cell response to cytotoxic damage is discussed. The mass spectrometry proteomics data ("raw", "mgf" and "xml" files) have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD007789 and PXD007776.

Changes in the Proteome of HaCaT Keratinocytes Induced by Cytotoxic Substance Triton X-100.

Changes in the proteome of keratinocytes of immortalized HaCaT line exposed to cytotoxic substance Triton X-100 in concentrations of 12.5 and 25 µg/ml were studied by liquid chromatography combined with mass spectrometry. The appearance of proteins involved in the regulation of mitosis, RNA stability, and catabolic processes were detected; the number of apoptosis-associated proteins increased, while the number of proteins involved in differentiation and energy metabolism of keratinocytes decreased.

Sodium Dodecyl Sulfate Cytotoxicity towards HaCaT Keratinocytes: Comparative Analysis of Methods for Evaluation of Cell Viability.

Viability of keratinocytes of HaCaT immortalized line incubated with sodium dodecyl sulfate for 3 min, 1 and 48 h, was studied by light microscopy, MTT test, and neutral red absorption test. The IC50 values were determined for each of the studied lengths of exposure. HaCaT cells exhibited a dose-dependent decrease of viability under the effect of sodium dodecyl sulfate, proportional to the length of exposure. The values measured by different methods (MTT test and neutral red absorption test) varied, the differences were determined by the duration of exposure to sodium dodecyl sulfate. The dispersion of values for 1 and 48 h exposure, obtained by MTT method, was greater than of the values obtained by neutral red absorption test.

Effects of cadmium chloride on the functional state of human intestinal cells.

Toxic effects of cadmium chloride in concentration range from 1 to 300 µM on differentiated human intestinal epithelial Caco-2 cells after three hours of exposure were investigated. Processes of disorganization of the actin cytoskeleton associated with the toxic effects of cadmium were characterized by fluorescent microscopy. The cadmium-induced activation of cellular stress response processes (changes in the mRNA expression of caspase-3, heat-shock and oxidative stress genes) has been demonstrated. The study revealed dose-dependent changes in mRNA expression levels of proteins involved in the formation of adherens (E-Cadherin and p120 catenin) and tight intercellular junction contacts (Claudin 4 and ZO1). The time- and concentration-dependent trend of cell monolayer transepithelial resistance lowering, characterizing the loss of intercellular contacts density with prolongation of cell exposure cadmium chloride was estimated. Results indicates that proteins associated with tight and adhesion junctions are primary targets of cadmium. Amongst genes involved in cell junction formation, the genes encoding E-Cadherin and p120-catenin proved to be the most sensitive to cadmium influence.

Expression of stress-dependent genes in hepatocytes spheroids after cisplatin treatment.

Effects of cisplatin on the hepatic HepaRG cells cultured in spheroids were estimated using biochemical, cytofluorometric, and molecular methods. Hepatocyte viability and expression of mRNA of stress-dependent genes involved in the cell response to toxic agent cisplatin underwent time- and dose-dependent changes. Activation of oxidative stress was observed at the early stages of incubation (3 h) followed by induction of apoptosis after prolonging the incubation to 24 h. The prospects of using HepaRG cells cultured in spheroids for estimation of drug toxicity by variations in the expression of stress-dependent genes were demonstrated. An increase in expression of genes of GSR and HSPA1A proteins at the early stages of incubation with cisplatin can serve as a marker of the cytotoxic effects of cisplatin and other agents with similar mechanisms of action.

[Genetically encoded FRET-pair on the basis of terbium-binding peptide and red fluorescent protein].

The genetically encoded FRET-pair was developed on the basis of terbium-binding peptide and red fluorescent protein DsRed2. To study fluorescence resonance energy transfer within FRET-pair, the gene-engineered construction was obtained, where sequences of terbium-binding peptide and red fluorescent protein DsRed2 were fused in single reading frame. The expression of this construction in strain E. coli BL21 (DE3) was studied and conditions of synthesis, isolation and purification of recombinant protein were optimized. The hydrodynamic radius of hybrid protein was determined by the method of dynamic light scattering. Energy transfer between sensitized terbium and red fluorescent protein was confirmed by the methods of phosporescent spectroscopy. The obtained FRET-pair can be used both for studies in vitro and as a reporter in living cells.