Tooth replacement in vertebrates depends on the persistence of the dental lamina, yet the molecular mechanisms that determine species-specific regenerative capacity remain poorly understood. Here we combine single-cell transcriptomics, lineage tracing, and genetic inactivation to define the role of LGR5, a canonical epithelial stem cell marker, in sequential molar development. We identify an unrecognized epithelial Lgr5⁺ population in the dental stalk and rudimentary successional dental lamina, together with a distinct mesenchymal Lgr5⁺ progenitor pool outside the tooth germ. Lineage tracing demonstrates that epithelial Lgr5⁺ cells contribute regionally to second molar formation, implicating them in sequential tooth initiation. Loss of Lgr5 disrupts lamina architecture, leading to shortened stalks, reduced SOX2 expression, impaired basement membrane integrity, and altered Wnt signaling, including downregulation of the LGR5-interacting protein PTK7. Organoid assays further show that Lgr5⁺ epithelial cells function as niche stabilizers rather than classical proliferative progenitors, providing structural and signaling support for replacement tooth development. Comparative analysis in diphyodont minipigs reveals conserved Lgr5 expression in non-regressing lamina domains, linking Lgr5 activity with species capable of multiple tooth generations. Together, these results identify Lgr5 as a key regulator of dental lamina stability and sequential tooth development, uncovering molecular mechanisms that couple Wnt signaling with epithelial integrity and establishing a framework for Lgr5-based regenerative strategies in dentistry.

Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Figure S1. In vitro profiling of SCH900776 and its selected analogs. Figure S2. Analogs of MU380 with the N-trifluoromethylpyrazol pharmacophore. Figure S3. Dot plots of flow cytometric analysis using γH2AX and Annexin V staining related to Figure 2D. Figure S4. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentrations in combination with HU in the concentration range shown on the x-axis (related to Figure 3A). Figure S5. Dynamics of cytotoxic effects of CHK1 inhibitors alone and in combination with HU. Figure S6. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentration in combination with GEM in concentration range shown on the x-axis (related to Figure 3B). Figure S7. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentration in combination with irradiation in the dose range shown on the x-axis. Figure S8. Western blot analysis of total CHK1 and p-CHK1 (S296) levels in a panel of selected cell lines after indicated treatments. Figure S9. Drug retention analysis. Figure S10. MU380 induces CHK1 pS345 in mice hair follicles. Figure S11. Bioluminescent signal of SHO mice bearing MiaPaCa2 luc xenografts treated with GEM, SCH900776, or MU380 alone or in indicated combinations. Weight of MiaPaCa2 luc tumor-bearing animals. Representative images of γH2AX staining in MiaPaCa2 luc tumor sections.

Table S1. Mouse plasma protein binding profile of MU380 and SCH900776. Table S2. IC50 determination of main P450 cytochrome isoforms in human liver microsomes. Table S3. A) Evaluation of MU380 and SCH900776 disappearance with time in the presence of human liver microsomes. B) Human microsomal stability intrinsic clearance data for MU380 and SCH900776. Table S4. Statistical analysis of dose response curves for cell lines indicated in the Figure 3A. Table S5. Statistical analysis of dose response curves for cell lines indicated in the Figure 3B. Table S6. Distribution properties of SCH900776 and MU380 obtained using Caco-2 permeability drug assay. Table S7. Pharmacokinetics of SCH900776 and MU380. Table S8. Residual kinase activities for SCH900776, MU380, and MU378 (1 μmol/L) measured in a panel of 207 human kinases (Eurofins KinaseProfiler service). Table S9. The median survival time. Table S10. Log-rank (Mantel-Cox) test, used to assess the statistical differences in survival curves among the groups. Table S11. Available data for mutational status of the cell lines included in the screen were extracted from Cancer Cell Line Encyclopedia (CCLE) database.

Correlative light and electron microscopy (CLEM) is an essential tool that allows for localisation of a particular target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometer scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of serial sections by both, single-molecule localization microscopy and transmission electron microscopy for correlative analysis. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows a dramatic improvement in fluorophore blinking behavior relative to the whole-mount approaches. Here we used commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as FASCIATA1 and NUCLEOLIN1 mutants and applied our on-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Detail description of synthesis and supporting data

Detail description of Material and Methods

Life-science experimental methods generate vast and ever-increasing volumes of data, which provide highly valuable research resources. However, management of these data is nontrivial and applicable software solutions are currently subject to intensive development. The solutions mainly fall into one of the two groups: general data management systems (e.g. Onedata, iRODS, B2SHARE, CERNBox) or very specialised data management solutions (e.g. solutions for biomolecular simulation data, biological imaging data, genomic data). To bridge this gap between them, we provide Onedata4Sci, a prototype data management solution, which is focused on the management of life science data and covers four key steps of the data life cycle, i.e. data acquisition, user access, computational processing and archiving. Onedata4Sci is based on the Onedata data management system. It is written in Python, fully containerised, with the support for processing the stored data in Kubernetes. The applicability of Onedata4Sci is shown in three distinct use cases -- plant imaging data, cellular imaging data, and cryo-electron microscopy data. Despite the use cases covering very different types of data and user patterns, Onedata4Sci demonstrated an ability to successfully handle all these conditions. Complete source codes of Onedata4Sci are available on GitHub (https://github.com/CERIT-SC/onedata4sci), and its documentation and manual for installation are also provided.

Supplementary Legends for Supplemetary Figure S1 - S11

Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5), a Wnt pathway member, has been previously recognised as a stem cell marker in numerous epithelial tissues. In this study, we used Lgr5-EGFP-CreERT2 mice to analyse the distribution of LGR5-positive cells during craniofacial development. LGR5 expressing cells were primarily located in the mesenchyme adjacent to the craniofacial epithelial structures undergoing folding, such as the nasopharyngeal duct, lingual groove, and vomeronasal organ. To follow the fate of LGR5-positive cells, we performed lineage tracing using an inducible Cre knock-in allele in combination with Rosa26-tdTomato reporter mice. The slight expansion of LGR5-positive cells was found around the vomeronasal organ, in the nasal cavity, and around the epithelium in the lingual groove. However, most LGR5 expressing cells remained in their original location, possibly supporting their signalling function for adjacent epithelium rather than exerting their role as progenitor cells for the craniofacial structures. Moreover, Lgr5 knockout mice displayed distinct defects in LGR5-positive areas, especially in the reduction of the nasopharyngeal duct, the alteration of the palatal shelves shape, abnormal epithelial folding in the lingual groove area, and the disruption of salivary gland development. The latter defect manifested as an atypical number and localisation of the glandular ducts. The gene expression of several Wnt pathway members (Rspo1-3, Axin2) was altered in Lgr5-deficient animals. However, the difference was not found in sorted EGFP-positive cells obtained from Lgr5 (+/+) and Lgr5 (-/-) animals. Expression profiling of LGR5-positive cells revealed the expression of several markers of mesenchymal cells, antagonists, as well as agonists, of Wnt signalling, and molecules associated with the basal membrane. Therefore, LGR5-positive cells in the craniofacial area represent a very specific population of mesenchymal cells adjacent to the epithelium undergoing folding or groove formation. Our results indicate a possible novel role of LGR5 in the regulation of morphogenetic processes during the formation of complex epithelial structures in the craniofacial areas, a role which is not related to the stem cell properties of LGR5-positive cells as was previously defined for various epithelial tissues.

Embryonic stem (ES) cells are pluripotent cells widely used in cell therapy and tissue engineering. However, the broader clinical applications of ES cells are limited by their genomic instability and karyotypic abnormalities. Thus, understanding the mechanisms underlying ES cell karyotypic abnormalities is critical to optimizing their clinical use. In this study, we focused on proliferating human and mouse ES cells undergoing multipolar divisions. Specifically, we analyzed the frequency and outcomes of such divisions using a combination of time-lapse microscopy and cell tracking. This revealed that cells resulting from multipolar divisions were not only viable, but they also frequently underwent subsequent cell divisions. Our novel data also showed that in human and mouse ES cells, multipolar spindles allowed more robust escape from chromosome segregation control mechanisms than bipolar spindles. Considering the frequency of multipolar divisions in proliferating ES cells, it is conceivable that cell division errors underlie ES cell karyotypic instability.

Centrioles account for centrosomes and cilia formation. Recently, a link between centrosomal components and human developmental disorders has been established. However, the exact mechanisms how centrosome abnormalities influence embryogenesis and cell fate are not understood. PLK4-STIL module represents a key element of centrosome duplication cycle. We analyzed consequences of inactivation of the module for early events of embryogenesis in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We demonstrate that blocking of PLK4 or STIL functions leads to centrosome loss followed by both p53-dependent and -independent defects, including prolonged cell divisions, upregulation of p53, chromosome instability, and, importantly, reduction of pluripotency markers and induction of differentiation. We show that the observed loss of key stem cells properties is connected to alterations in mitotic timing and protein turnover. In sum, our data define a link between centrosome, its regulators, and the control of pluripotency and differentiation in PSCs.

Extracellular matrix (ECM) forms an essential part of tissue microenvironment. Compositional and structural properties of ECM modulate behaviour of cells, including their differentiation, proliferation, and turnover. Here we aimed at detailed characterization of the nanostructure of lung ECM and evaluation of its bioactivity on cells grown in vitro. Mouse lungs were decellularized using 0.2 % sodium dodecyl sulphate, hypotonic solutions, and DNase. Morphological analysis of the resulting ECM scaffolds was performed by means of transmission and scanning electron microscopy. The ECM scaffolds retained 3D architecture of the main lung anatomical regions: the alveolar region and the blood/ airway network. The region-specific ECM nanotopology and organization of ECM macromolecules such as fibres of collagen, elastin, and fibrillin were characterized. The lung ECM scaffolds were also homogenized and applied as a supplement to growth medium on primary lung cells in vitro to test the bioactivity of lung ECM. We demonstrate that homogenized ECM does not have a negative impact on the proliferation rate of primary lung cells. In conclusion, we herein show at nanoscale the morphological characteristics of lung ECM and demonstrate that lung ECM produced by decellularization procedure is compatible with in vitro cultured lung cells. These findings add to a better understanding of cells with their natural environment and can be valuable when designing applications of ECM for purposes in tissue engineering.

HMGB1 and HMGB2 proteins have been implicated in numerous cellular processes, including proliferation, differentiation, apoptosis, and tumor growth. It is unknown whether they are involved in regulating the typical functions of pluripotent human embryonic stem cells (hESCs) and/or those of the differentiated derivatives of hESCs. Using inducible, stably transfected hESCs capable of shRNA-mediated knockdown of HMGB1 and HMGB2, we provide evidence that downregulation of HMGB1 and/or HMGB2 in undifferentiated hESCs does not affect the stemness of cells and induces only minor changes to the proliferation rate, cell-cycle profile, and apoptosis. After differentiation is induced, however, the downregulation of those proteins has important effects on proliferation, apoptosis, telomerase activity, and the efficiency of differentiation toward the neuroectodermal lineage. Furthermore, those processes are affected only when one, but not both, of the two proteins is downregulated; the knockdown of both HMGB1 and HMGB2 results in a normal phenotype. Those results advance our knowledge of regulation of hESC and human neuroectodermal cell differentiation and illustrate the distinct roles of HMGB1 and HMGB2 during early human development.

Abstract Checkpoint-mediated dependency of tumor cells can be deployed to selectively kill them without substantial toxicity to normal cells. Specifically, loss of CHK1, a serine threonine kinase involved in the surveillance of the G2–M checkpoint in the presence of replication stress inflicted by DNA-damaging drugs, has been reported to dramatically influence the viability of tumor cells. CHK1′s pivotal role in maintaining genomic stability offers attractive opportunity for increasing the selectivity, effectivity, and reduced toxicity of chemotherapy. Some recently identified CHK1 inhibitors entered clinical trials in combination with DNA antimetabolites. Herein, we report synthesis and profiling of MU380, a nontrivial analogue of clinically profiled compound SCH900776 possessing the highly unusual N-trifluoromethylpyrazole motif, which was envisioned not to undergo metabolic oxidative dealkylation and thereby provide greater robustness to the compound. MU380 is a selective and potent inhibitor of CHK1 which sensitizes a variety of tumor cell lines to hydroxyurea or gemcitabine up to 10 times. MU380 shows extended inhibitory effects in cells, and unlike SCH900776, does not undergo in vivo N-dealkylation to the significantly less selective metabolite. Compared with SCH900776, MU380 in combination with GEM causes higher accumulation of DNA damage in tumor cells and subsequent enhanced cell death, and is more efficacious in the A2780 xenograft mouse model. Overall, MU380 represents a novel state-of-the-art CHK1 inhibitor with high potency, selectivity, and improved metabolic robustness to oxidative N-dealkylation. Mol Cancer Ther; 16(9); 1831–42. ©2017 AACR.

Fanconi anemia (FA) is a rare genetic disorder associated with bone-marrow failure, genome instability and cancer predisposition. Recently, we and others have demonstrated dysfunctional mitochondria with morphological alterations in FA cells accompanied by high reactive oxygen species (ROS) levels. Mitochondrial morphology is regulated by continuous fusion and fission events and the misbalance between these two is often accompanied by autophagy. Here, we provide evidence of impaired autophagy in FA. We demonstrate that FA cells have increased number of autophagic (presumably mitophagic) events and accumulate dysfunctional mitochondria due to an impaired ability to degrade them. Moreover, mitochondrial fission accompanied by oxidative stress (OS) is a prerequisite condition for mitophagy in FA and blocking this pathway may release autophagic machinery to clear dysfunctional mitochondria.

A significant part of current research studies utilizes various cellular models which imply specific antibiotics-containing media as well as antibiotics used for clonal selection or promoter de/activation. With the great success of developing such tools, mitochondria, once originated from bacteria, can be effectively targeted by antibiotics. For that reason, some studies propose antibiotics-targeting of mitochondria as part of anticancer therapy. Here, we have focused on the effects of various classes of antibiotics on mitochondria in cancer and non-cancer cells and demonlow mitochondrial membrane potential, reduced ATP production, altered morphology and lowered respiration rate which altogether suggested mitochondrial dysfunction (MDF). This was in parallel with increased level of reactive oxygen species (ROS) and decreased activity of mitochondrial respiration complexes. However, both survival and repopulation capacity of cancer cells was not significantly affected by the antibiotics, perhaps due to a glycolytic shift or activated autophagy. In turn, simultaneous inhibition of autophagy and treatment with antibiotics largely reduced tumorigenic properties of cancer cells suggesting potential strategy for anticancer therapy.

Ovarian cancer is one of the most common malignancies in women and contributes greatly to cancer‐related deaths. Tumor suppressor candidate 3 (TUSC3) is a putative tumor suppressor gene located at chromosomal region 8p22, which is often lost in epithelial cancers. Epigenetic silencing of TUSC3 has been associated with poor prognosis, and hypermethylation of its promoter provides an independent biomarker of overall and disease‐free survival in ovarian cancer patients. TUSC3 is localized to the endoplasmic reticulum in an oligosaccharyl tranferase complex responsible for the N‐glycosylation of proteins. However, the precise molecular role of TUSC3 in ovarian cancer remains unclear. In this study, we establish TUSC3 as a novel ovarian cancer tumor suppressor using a xenograft mouse model and demonstrate that loss of TUSC3 alters the molecular response to endoplasmic reticulum stress and induces hallmarks of the epithelial‐to‐mesenchymal transition in ovarian cancer cells. In summary, we have confirmed the tumor‐suppressive function of TUSC3 and identified the possible mechanism driving TUSC3‐deficient ovarian cancer cells toward a malignant phenotype.