ABSTRACT The macrolide antibiotic, macrolactin A (McA), has been known for its antimicrobial properties since the late 1980s, but the mechanism of its antibacterial activity is still unknown. In this study, we investigated the microbiological and molecular characteristics of McA antimicrobial activity. McA effect on bacteria was found to be both bacteriostatic and bactericidal, depending on species and strains. Regarding the mechanism of action of McA, the following important results were obtained: 1) using in vivo and in vitro systems, we showed that McA is an inhibitor of protein synthesis in bacteria; 2) the concentration of McA required to inhibit protein synthesis in the E. coli cell-free model was found to be 50 times lower than the concentration required in the S. aureus cell-free model; 3) the toe-printing assay revealed that McA inhibits the first step of elongation stage of protein synthesis; 4) we identified single and multiple nucleotide polymorphisms in the gene encoding the translation elongation factor Tu (EF-Tu) by annotating the genomes of McA-resistant Bacillus pumilus McA R and its parental strain. Molecular modeling showed that the McA molecule can form non-covalent bonds with amino acids at the interface of domains 1 and 2 of EF-Tu, characterized by a relatively high docking score. Overall, our study demonstrated that McA acts as an elfamycin-like antibiotic (targeting EF-Tu), addressing a substantial gap in our understanding of the mechanism of action of macrolactin A, a representative member of macrolides.

In recent decades, there has been a significant amount of research on the biological role of non-coding RNAs (ncRNAs) in both normal and pathological conditions. Specifically, a growing body of evidence suggests that ncRNAs play a crucial role in maintaining epidermal homeostasis. These ncRNAs are involved in regulating epidermal differentiation and wound healing, as well as in pathological skin conditions, such as psoriasis and chronic wounds. The discovery of mechanisms such as RNA interference and other modes of action of ncRNAs has led to the development of novel therapeutic strategies, where ncRNAs could serve as targets, therapeutic agents, or diagnostic markers. This review explores the role of different classes of ncRNAs in the epidermis under normal and abnormal conditions, the mechanisms by which ncRNAs interact with other modulators of epidermal homeostasis, and the current state of ncRNA-based therapy.

The aryl hydrocarbon receptor (AhR) plays a crucial role in mediating xenobiotic responses, as well as regulating broader metabolic, differentiation, and stress response programs. In this study, we present a comprehensive long-read RNA sequencing dataset that examines transcriptional changes in the HepaRG human cell line during differentiation induced by dimethyl sulfoxide (DMSO) and acute activation of the AhR with 3-methylcholanthrene (3-MC). We identified 946 genes that were differentially expressed between the NonDiff and Diff conditions (303 genes upregulated and 643 genes downregulated), and 1786 genes that showed differential expression between Diff and Ind conditions (961 genes upregulated and 825 genes downregulated). The acute induction of 3-MC produced a robust AhR signature, characterized by the robust induction of CYP1A1 and CYP1B1, along with a coordinated downregulation of several constitutive hepatic genes involved in drug metabolism (e.g., CYP3A4 and CYP2C8). To facilitate further analysis and reuse of our data, we have provided processed gene-level count matrices, transcript per million (TPM) tables, and detailed differential expression results, as well as analysis scripts. This resource supports research into AhR biology, pharmacogene regulation, and the development of methods for long-read transcriptomics in liver models.

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.

Keratins 6, 16, and 17 occupy unique positions within the keratin family. These proteins are not commonly found in the healthy, intact epidermis, but their expression increases in response to damage, inflammation, and hereditary skin conditions, as well as cancerous cell transformations and tumor growth. As a result, there is an active investigation into the potential use of these proteins as biomarkers for different pathologies. Recent studies have revealed the role of these keratins in regulating keratinocyte migration, proliferation, and growth, and more recently, their nuclear functions, including their role in maintaining nuclear structure and responding to DNA damage, have also been identified. This review aims to summarize the latest research on keratins 6, 16, and 17, their regulation in the epidermis, and their potential use as biomarkers in various skin conditions.

Approximately 50% of tumors carry mutations in TP53; thus, evaluation of the features of mutant p53 is crucial to understanding the mechanisms underlying cell transformation and tumor progression. HaCaT keratinocytes represent a valuable model for research in this area since they are considered normal, although they bear two gain-of-function mutations in TP53. In the present study, transcriptomic and proteomic profiling were employed to examine the functions of mutant p53 and to investigate the impact of its complete abolishment. Our findings indicate that CRISPR-mediated TP53 knockout results in significant changes at the transcriptomic and proteomic levels. The knockout of TP53 significantly increased the migration rate and altered the expression of genes associated with invasion, migration, and EMT but suppressed the epidermal differentiation program. These outcomes suggest that, despite being dysfunctional, p53 may still possess oncosuppressive functions. However, despite being considered normal keratinocytes, HaCaT cells exhibit oncogenic properties.

Background: Cadmium exposure induces dermatotoxicity and epidermal barrier disruption and leads to the development of various pathologies. HaCaT cells are immortalized human keratinocytes that are widely used as alternatives to primary human keratinocytes, particularly for evaluating cadmium toxicity. HaCaT cells bear two gain-of-function (GOF) mutations in the TP53 gene, which strongly affect p53 function. Mutant forms of p53 are known to correlate with increased resistance to various stimuli, including exposure to cytotoxic substances. In addition, keratin 17 (KRT17) was recently shown to be highly expressed in HaCaT cells in response to genotoxic stress. Moreover, p53 is a direct transcriptional repressor of KRT17. However, the impact of TP53 mutations in HaCaT cells on the regulation of cell death and keratin 17 expression is unclear. In this study, we aimed to evaluate the impact of p53 on the response to Cd-induced cytotoxicity Methods and results: Employing the MTT assay and Annexin V/propidium iodide staining, we demonstrated that knockout of TP53 leads to a decrease in the sensitivity of HaCaT cells to the cytotoxic effects of cadmium. Specifically, HaCaT cells with TP53 knockout (TP53 KO HaCaT) exhibited cell death at a cadmium concentration of 10 mu M or higher, whereas wild-type cells displayed cell death at a concentration of 30 mu M. Furthermore, apoptotic cells were consistently detected in TP53 KO HaCaT cells upon exposure to low concentrations of cadmium (10 and 20 mu M) but not in wild-type cells. Our findings also indicate that cadmium cytotoxicity is mediated by reactive oxygen species (ROS), which were significantly increased only in TP53 knockout cells treated with 30 mu M cadmium. An examination of proteomic data revealed that TP53 knockout in HaCaT cells resulted in the upregulation of proteins involved in the regulation of apoptosis, redox systems, and DNA repair. Moreover, RT-qPCR and immunoblotting showed that cadmium toxicity leads to dose-dependent induction of keratin 17 in p53-deficient cells but not in wild-type cells. Conclusions: The connection between mutant p53 in HaCaT keratinocytes and increased resistance to cadmium toxicity was demonstrated for the first time. Proteomic profiling revealed that TP53 knockout in HaCaT cells led to the activation of apoptosis regulatory circuits, redox systems, and DNA repair. In addition, our data support the involvement of keratin 17 in the regulation of DNA repair and cell death. Apparently, the induction of keratin 17 is p53-independent but may be inhibited by mutant p53.

An increasing number of studies have investigated the effects of Cd on human health. Cd-induced dermatotoxicity is an important field of research, but numerous studies have focused on the effects of Cd on the human skin. Moreover, most studies have been performed using HaCaT cells but not primary keratinocytes. In this study, we provide the results describing the cytotoxic effects of Cd exposure on primary human epidermal keratinocytes obtained from different donors. The subtoxic concentration of cadmium chloride was determined via MTT assay, and transcriptomic analysis of the cells exposed to this concentration (25 µM) was performed. As in HaCaT cells, Cd exposure resulted in increased ROS levels, cell cycle arrest, and induction of apoptosis. In addition, we report that exposure to Cd affects zinc and copper homeostasis, induces metallothionein expression, and activates various signaling pathways, including Nrf2, NF-kB, TRAIL, and PI3K. Cd induces the secretion of various cytokines (IL-1, IL-6, IL-10, and PGE2) and upregulates the expression of several cytokeratins, such as KRT6B, KRT6C, KRT16, and KRT17. The results provide a better understanding of the mechanisms of cadmium-induced cytotoxicity and its effect on human epidermal skin cells.

Background: Alzheimer’s disease (AD) develops as a result of oxidative damage to neurons and chronic inflammation of microglia. These processes can be influenced by the use of a conditioned medium (CM) derived from mesenchymal stem cells (MSCs). The CM contains a wide range of factors that have neurotrophic, antioxidant, and anti-inflammatory effects. In addition, the therapeutic potential of the CM can be further enhanced by pretreating the MSCs to increase their paracrine activity. The current study aimed to investigate the neuroprotective effects of CM derived from MSCs, which were either activated by a TLR3 ligand or exposed to CoCl2, a hypoxia mimetic (pCM or hCM, respectively), in an in vitro model of AD. Methods: We have developed a novel in vitro model of AD that allows us to investigate the neuroprotective and anti-inflammatory effects of MSCs on induced neurodegeneration in the PC12 cell line and the activation of microglia using THP-1 cells. Results: This study demonstrates for the first time that pCM and hCM exhibit more pronounced immunosuppressive effects on proinflammatory M1 macrophages compared to CM derived from untreated MSCs (cCM). This may help prevent the development of neuroinflammation by balancing the M1 and M2 microglial phenotypes via the decreased secretion of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and increased secretion of IL-4, as well as the expression of IL-10 and TGF-β by macrophages. Moreover, a previously unknown increase in the neurotrophic properties of hCM was discovered, which led to an increase in the viability of neuron-like PC12 cells under H2O2-induced oxidative-stress conditions. These results are likely associated with an increase in the production of growth factors, including vascular endothelial growth factor (VEGF). In addition, the neuroprotective effects of CM from preconditioned MSCs are also mediated by the activation of the Nrf2/ARE pathway in PC12 cells. Conclusions: TLR3 activation in MSCs leads to more potent immunosuppressive effects of the CM against pro-inflammatory M1 macrophages, while the use of hCM led to increased neurotrophic effects after H2O2-induced damage to neuronal cells. These results are of interest for the potential treatment of AD with CM from preactivated MSCs.

Combining new therapeutics with all-trans-retinoic acid (ATRA) could improve the efficiency of acute myeloid leukemia (AML) treatment. Modeling the process of ATRA-induced differentiation based on the transcriptomic profile of leukemic cells resulted in the identification of key targets that can be used to increase the therapeutic effect of ATRA. The genome-scale transcriptome analysis revealed the early molecular response to the ATRA treatment of HL-60 cells. In this study, we performed the transcriptomic profiling of HL-60, NB4, and K562 cells exposed to ATRA for 3-72 h. After treatment with ATRA for 3, 12, 24, and 72 h, we found 222, 391, 359, and 1032 differentially expressed genes (DEGs) in HL-60 cells, as well as 641, 1037, 1011, and 1499 DEGs in NB4 cells. We also found 538 and 119 DEGs in K562 cells treated with ATRA for 24 h and 72 h, respectively. Based on experimental transcriptomic data, we performed hierarchical modeling and determined cyclin-dependent kinase 6 (CDK6), tumor necrosis factor alpha (TNF-alpha), and transcriptional repressor CUX1 as the key regulators of the molecular response to the ATRA treatment in HL-60, NB4, and K562 cell lines, respectively. Mapping the data of TMT-based mass-spectrometric profiling on the modeling schemes, we determined CDK6 expression at the proteome level and its down-regulation at the transcriptome and proteome levels in cells treated with ATRA for 72 h. The combination of therapy with a CDK6 inhibitor (palbociclib) and ATRA (tretinoin) could be an alternative approach for the treatment of acute myeloid leukemia (AML).

Dlya sertifikacii novyh farmacevticheskih, himicheskih ili kosmeticheskih produktov neobhodimo testirovanie na toksichnost', v tom chisle dermatotoksichnost'. Modeli in vitro issledovanij schitayut naibolee perspektivnymi, i ryad takih testov vnedren v praktiku sertifikacionnyh ispytanij. Novye vozmozhnosti dlya registracii rannego otveta kletok na povrezhdenie mogut predostavit' kletochnye sensory, osnovannye na vizual'noj kolichestvennoj registracii izmenenij aktivnosti signal'nyh putej, zadejstvovannyh v realizacii takogo otveta. NF-κB i AP-1 — dva vazhnyh faktora transkripcii belkov, aktivnost' kotoryh vozrastaet v kletke pri povrezhdenii, vospalenii i izmenenii redoks-balansa. Cel'yu issledovaniya bylo razrabotat' kletochnye sensory na osnove immortalizovannyh keratinocitov cheloveka linii HaCaT, kotorye ekspressiruyut zelenyj fluorescentnyj belok (GFP) pri aktivacii signal'nyh putej NF-κB (HaCaT/NF-κB) ili AP-1 (HaCaT/AP-1), i izuchit' ih informativnost' pri registracii dozozavisimogo otveta na vozdejstvie induktorov sootvetstvuyushchih signal'nyh putej. Rezul'taty pokazali, chto uroven' fluorescencii kletok HaCaT/NF-κB dozozavisimo izmenyalsya v 6,05 ± 0,51 i 5,53 ± 0,52 raz pri vozdejstvii TNFα ili LPS (v koncentraciyah ot 0 do 80 ng/ml). Biosensor HaCaT/AP-1 takzhe reagiroval na vozdejstvie Cd (NO3)2 (v koncentraciyah ot 0 do 40 mkM) i ul'trafioletovogo izlucheniya tipa A (UF-A) (ot 0 do 40 Dzh/sm2), odnako pozvolyal registrirovat' ego kachestvenno, no ne kolichestvenno. Fluorescenciya kletok sensora vozrastala v 1,51 ± 0,24 i 1,66 ± 0,43 raz sootvetstvenno. Razrabotannye kletochnye sensory mogut byt' ispol'zovany dlya ocenki citotoksicheskogo dejstviya testiruemyh veshchestv na kletki kozhi cheloveka in vitro i izucheniya mekhanizmov citotoksichnosti.

Using tandem mass spectrometry with electrospray ionization, a comparative analysis of HaCaT keratinocyte proteins was carried out before and after exposure of cells to sodium dodecyl sulfate (25 mg/ml) for 48 hours; proteins encoded by human chromosome 18 genes were chosen as the comparison proteins. A total of 2418 proteins were detected in the HaCaT immortalized human keratinocytes, 70% of these proteins were identified by two or more unique peptides. Panoramic mass spectrometry analysis identified 38 proteins encoded by chromosome 18 genes, 27 proteins were common to control HaCaT cells and HaCaT cells exposed to SDS. Using the Metascape database (https://metascape.org), an enrichment analysis of GO terms of the Biological Process category of chromosome 18 gene encoded proteins of HaCaT keratinocytes was performed before and after the SDS exposure. The SDS exposure resulted in a slight enrichment of the GO term "response to stimulus" (GO:0050896) and the related GO term "negative regulation of biological process" (GO:0048519). We found decreased expression levels of membrane proteins encoded by chromosome 18 genes related to cell-cell adhesion (GO:0098609), such as DSC1, DSC3, and DSG1. A decrease in the expression level of desmosomal cadherins is characteristic of malignant neoplasms developing from epithelial tissue cells of various internal organs, mucous membranes, and skin. The method of preparation of HaCaT keratinocyte samples used in this work increased the sensitivity of proteomic analysis of cell culture and made it possible to identify twice as many proteins in one gel strip as compared to the number of proteins (1284) in HaCaT samples subjected to osmotic shock and cleavage by trypsin in solution.

Background Mesenchymal stromal cells (MSCs) have regenerative and immunomodulatory properties, making them suitable for cell therapy. Toll-like receptors (TLRs) in MSCs respond to viral load by secreting immunosuppressive or proinflammatory molecules. The expression of anti-inflammatory molecules in MSCs can be altered by the concentration and duration of exposure to the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)). This study aimed to optimize the preconditioning of MSCs with poly(I:C) to increase immunosuppressive effects and to identify MSCs with activated TLR3 (prMSCs). Methods Flow cytometry and histochemical staining were used to analyze MSCs for immunophenotype and differentiation potential. MSCs were exposed to poly(I:C) at 1 and 10 μg/mL for 1, 3, and 24 h, followed by determination of the expression of IDO1 , WARS1 , PD-L1 , TSG-6 , and PTGES2 and PGE2 secretion. MSCs and prMSCs were cocultured with intact (J − ) and activated (J + ) Jurkat T cells. The proportion of proliferating and apoptotic J + and J − cells, IL-10 secretion, and IL-2 production after cocultivation with MSCs and prMSCs were measured. Liquid chromatography–mass spectrometry and bioinformatics analysis identified proteins linked to TLR3 activation in MSCs. Results Poly(I:C) at 10 μg/mL during a 3-h incubation caused the highest expression of immunosuppression markers in MSCs. Activation of prMSCs caused a 18% decrease in proliferation and a one-third increase in apoptotic J + cells compared to intact MSCs. Cocultures of prMSCs and Jurkat cells had increased IL-10 and decreased IL-2 in the conditioned medium. A proteomic study of MSCs and prMSCs identified 53 proteins with altered expression. Filtering the dataset with Gene Ontology and Reactome Pathway revealed that poly(I:C)-induced proteins activate the antiviral response. Protein‒protein interactions by String in prMSCs revealed that the antiviral response and IFN I signaling circuits were more active than in native MSCs. prMSCs expressed more cell adhesion proteins (ICAM-I and Galectin-3), PARP14, PSMB8, USP18, and GBP4, which may explain their anti-inflammatory effects on Jurkat cells. Conclusions TLR3 activation in MSCs is dependent on exposure time and poly(I:C) concentration. The maximum expression of immunosuppressive molecules was observed with 10 µg/mL poly(I:C) for 3-h preconditioning. This priming protocol for MSCs enhances the immunosuppressive effects of prMSCs on T cells.

Liniya HaCaT — spontanno immortalizovannye nekancerogennye keratinocity cheloveka, shiroko ispol'zuemye v kachestve modeli dlya izucheniya funkcij normal'nyh keratinocitov cheloveka. V genome kletok HaCaT prisutstvuyut dve alleli gena TP53, kotorye soderzhat dve gain-of-function (GOV) mutacii, priobretennye v rezul'tate spontannoj immortalizacii (mutTP53). Nalichie mutacij privodit k uvelicheniyu skorosti proliferacii i narusheniyu programmy stratifikacii. Cel'yu issledovaniya bylo izuchit' vliyanie nokauta gena mutTR53 na sposobnost' keratinocitov linii HaCaT k proliferacii i migracii v modeli povrezhdeniya i regeneracii epidermisa in vitro (skretch-test), a takzhe na sposobnost' formirovat' mnogoslojnyj epitelij v organotipicheskoj modeli epidermisa. Dlya provedeniya skretch-testa kletki kul'tivirovali do obrazovaniya monosloya, zatem nanosili standartizovannoe povrezhdenie. Organotipicheskuyu model' poluchali kul'tivirovaniem keratinocitov v polikarbonatnyh membrannyh vstavkah s diametrom por 0,4 mkm na granice razdela faz (vozduh-zhidkost'). Prodemonstrirovano, chto nokaut mutantnogo gena TP53 privodit k uvelicheniyu migracionnoj sposobnosti keratinocitov linii HaCaT: dlya HaCaT s nokautom mutTP53 zakrytie defekta proiskhodilo bystree po sravneniyu s sootvetstvuyushchej gruppoj WT HaCaT (p < 0,05), na tret'i sutki razmer defekta sostavlyal 12% ± 3% i 66% ± 5% ot pervonachal'nogo. Polucheny dannye, chto mutantnyj TP53 v kletkah HaCaT yavlyaetsya negativnym regulyatorom ekspressii laminina-5 (ekspressiya LAMC2 byla vyshe v kletkah s nokautom mutTP53 v 9,96 ± 1,92 raz, p < 0,05), odnako eto ne sposobstvuet normalizacii programmy differencirovki i stratifikacii epiteliya s obrazovaniem rogovogo sloya v organotipicheskoj modeli.

There is no direct evidence supporting that SDS is a carcinogen, so to investigate this fact, we used HaCaT keratinocytes as a model of human epidermal cells. To reveal the candidate proteins and/or pathways characterizing the SDS impact on HaCaT, we proposed comparative proteoinformatics pipeline. For protein extraction, the performance of two sample preparation protocols was assessed: 0.2% SDS-based solubilization combined with the 1DE-gel concentration (Protocol 1) and osmotic shock (Protocol 2). As a result, in SDS-exposed HaCaT cells, Protocol 1 revealed 54 differentially expressed proteins (DEPs) involved in the disease of cellular proliferation (DOID:14566), whereas Protocol 2 found 45 DEPs of the same disease ID. The 'skin cancer' term was a single significant COSMIC term for Protocol 1 DEPs, including those involved in double-strand break repair pathway (BIR, GO:0000727). Considerable upregulation of BIR-associated proteins MCM3, MCM6, and MCM7 was detected. The eightfold increase in MCM6 level was verified by reverse transcription qPCR. Thus, Protocol 1 demonstrated high effectiveness in terms of the total number and sensitivity of MS identifications in HaCaT cell line proteomic analysis. The utility of Protocol 1 was confirmed by the revealed upregulation of cancer-associated MCM6 in HaCaT keratinocytes induced by non-toxic concentration of SDS. Data are available via ProteomeXchange with identifier PXD035202.

The immortalized keratinocytes, HaCaT, are a popular model for skin research (toxicity, irritation, allergic reactions, or interaction of cells). They maintain a stable keratinocyte phenotype and respond to keratinocyte differentiation stimuli. However, programs of stratification and expression of differentiation markers in HaCaT keratinocytes are aberrant. In HaCaT cells, there are two mutant p53 alleles (i.e., R282Q and H179Y) that contain gain-of-function (GOF) mutations resulting from spontaneous immortalization (mutp53). At the same time, mutp53 acts as a transcription factor and also affects the interaction of p63 protein with its transcription targets. Proteins of the p53 family are crucial for regulation of proliferation and differentiation processes in human keratinocytes, although the involvement of mutp53 in these processes is not fully clear. We present data sets obtained as a result of high-performance proteomic analysis of immortalized HaCaT keratinocytes with p53 knockout in two different states, subconfluent and confluent, which are characterized by different intensites of cell differentiation processes. To obtain the proteomic profiles of the cells, we applied LC-MS/MS measurements processed with MaxQuant software (version 1.6.3.4).

The HaCaT cell line represents the spontaneously immortalized non-carcinogenic human keratinocytes that are used as a model for studying the function of normal human keratinocytes. There are two TP53 alleles in the HaCaT cell genome, which comprise two gain-of-function (GOF) mutations acquired through spontaneous immortalization (mutTP53). Mutations result in the increased proliferation rate and violation of the stratification program. The study was aimed to assess the effects of the mutTP53 gene knockout on the HaCaT keratinocytes capability of proliferation and migration in the in vitro model of epidermal injury and regeneration (scratch test), and on the ability to form stratified epithelium in the organotypic epidermal model. To perform the scratch-test, cells were cultured until monolayer was formed, then the standardized wound was created. The organotypic model was obtained by growing keratinocytes in the polycarbonate membrane inserts with the pore size of 0.4 mu m at the interface between the phases (air-liquid). It has been shown that the mutant TP53 gene knockout results in the increased migration capability of the HaCaT keratinocytes: in the mutTP53 knockout HaCaT, the defect closure occurred faster than in the appropriate group of the WT HaCaT (p < 0.05), on day three the defect size was 12% +/- 3% and 66% +/- 5% of the initial size. There is evidence that mutant TP53 in the HaCaT cells is a negative regulator of the laminin-5 expression (LAMC2 expression was 9.96 +/- 1.92 times higher in the cells with the mutTP53 knockout, p < 0.05), however, this does not promote normalization of the program of epithelial differentiation and stratification followed by formation of the stratum corneum in the organotypic model.

The HaCaT line of immortalized non-tumor cells is a popular model of keratinocytes used for dermatological studies, in the practice of toxicological tests, and in the study of skin allergic reactions. These cells maintain a stable keratinocyte phenotype, do not require specific growth factors during cultivation, and respond to keratinocyte differentiation stimuli. HaCaT cells bear two mutant p53 alleles - R282Q and H179Y. At least two mechanisms of GOF (gain-of-function) of mutant p53 are known: it affects functions of p63/p73 by inhibiting their binding to DNA; or it binds to new DNA sites by interacting with other transcription factors (NF-Y, E2F1, NF-KB, VDR, p63). Proteins of the P53 family play an important role in the regulation of proliferation and differentiation processes of human keratinocytes. Proteomic study of HaCaT cells with TP53 gene knockdown provides new data for understanding the limitations of HaCaT cells when using them as an experimental model of normal human keratinocytes. In this article we present datasets obtained through the high-throughput shotgun proteomics analysis of human immortalized HaCaT keratinocytes and p53 knockdown 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 MaxQuant software (version 1.6.3.4). The "RAW" files were deposited to the ProteomeXchange with identifier PXD033538.

Studies of induced granulocytic differentiation help to reveal molecular mechanisms of cell maturation. The nuclear proteome represents a rich source of regulatory molecules, including transcription factors (TFs). It is important to have an understanding of molecular perturbations at the early stages of the differentiation processes. By applying the proteomic quantitative profiling using isobaric labeling, we found that the contents of 214, 319, 376, 426, and 391 proteins were altered at 3, 6, 9, 12, and 72 h, respectively, compared to 0 h in the HL-60 cell nuclear fraction under all-trans-retinoid acid (ATRA) treatment. From 1860 identified nuclear proteins, 231 proteins were annotated as proteins with transcription factor (TF) activity. Six TFs (RREB1, SRCAP, CCDC124, TRIM24, BRD7, and BUD31) were downregulated and three TFs EWSR1, ENO1, and FUS were upregulated at early time points (3–12 h) after ATRA treatment. Bioinformatic annotation indicates involvement of the HL-60 nuclear proteome in DNA damage recognition in the RUNX1-triggered pathway, and in the p53-regulation pathway. By applying scheduled multiple reaction monitoring using stable isotopically labeled peptide standards (MRM/SIS), we found a persistent increase in the content of the following proteins: PRAM1, CEPBP, RBPJ, and HIC1 in the HL-60 cell nuclear fraction during ATRA-induced granulocytic differentiation. In the case of STAT1, CASP3, PARP1, and PRKDC proteins, a transient increase in their content was observed at early time points (3–12 h) after the ATRA treatment. Obtained data on nuclear proteome composition and dynamics during granulocytic differentiation could be beneficial for the development of new treatment approaches for leukemias with the mutated p53 gene.

The HaCaT cell line represents the spontaneously immortalized non-carcinogenic human keratinocytes that are used as a model for studying the function of normal human keratinocytes. There are two TP53 alleles in the HaCaT cell genome, which comprise two gain-of-function (GOF) mutations acquired through spontaneous immortalization (mutTP53). Mutations result in the increased proliferation rate and violation of the stratification program. The study was aimed to assess the effects of the mutTP53 gene knockout on the HaCaT keratinocytes capability of proliferation and migration in the in vitro model of epidermal injury and regeneration (scratch test), and on the ability to form stratified epithelium in the organotypic epidermal model. To perform the scratch-test, cells were cultured until monolayer was formed, then the standardized injury was created. The organotypic model was obtained by growing keratinocytes in the polycarbonate membrane inserts with the pore size of 0.4 μm at the interface between the phases (air-liquid). It has been shown that the mutant TP53 gene knockout results in the increased migration capability of the HaCaT keratinocytes: in the HaCaT with the mutTP53 knockout, the defect closure occurred faster than in the appropriate group of the WT HaCaT (p < 0.05), on day three the defect size was 12% ± 3% and 66% ± 5% of the initial size. There is evidence that mutant TP53 in the HaCaT cells is a negative regulator of the laminin 5 expression (LAMC2 expression was 9.96 ± 1.92 times higher in the cells with the mutTP53 knockout, p < 0.05), however, this does not promote normalization of the program of epithelial differentiation and stratification followed by formation of the stratum corneum in the organotypic model.