High-content Imaging Research Articles

Selection of Force Sensors for In Situ Measurement of Neotissue Microenvironments.

Mechanical forces are a critical stimulus in both native and engineered tissues. Direct measurement of these microenvironmental forces has been challenging, particularly for cell-dense models. To address this, we previously developed hydrogel-based force sensors that are approximately the size of a cell and can be imaged over time to computationally assess the forces exerted by surrounding cells and matrix. The goal of this project was to identify how the physical characteristics of force sensors impact measurements. Sensors were varied in size, elastic modulus, and surface coating before being included in stem cell suspensions that then spontaneously self-assembled into spheroidal neotissues. Using this model of early mesenchymal condensation, we hypothesized that larger, softer sensors would provide greater sensitivity and precision, whereas protein coatings would influence the directionality of applied forces (tensile vs. compressive). These experiments were conducted using a high-content imaging system that allowed analysis of over a thousand sensors to evaluate the various conditions. Results indicated that measurement fidelity was highest for force sensors that had a diameter >20 µm and modulus ∼0.2 kPa. Extremely soft sensors deformed too much, whereas stiffer sensors deformed too little. Collagen and N-cadherin coatings, which replicated cell-matrix or cell-cell binding, respectively, allowed for tensile forces to be exerted on the sensors, with greater forces being observed for N-cadherin sensors in these highly cellular neotissue constructs. Uncoated sensors were universally compressed due to the lack of cell-sensor adhesion. Disruption of the actin cytoskeleton lessened microenvironmental forces, whereas disruption of microtubules had no measurable effect. Potential future applications of the technology include studies of in situ forces in developing tissues as well as a real-time sensor for monitoring the growth of engineered constructs.

SMCVdb: a database of experimental cellular toxicity information for drug candidate molecules.

Many drug discovery exercises fail because small molecules that are effective inhibitors of target proteins exhibit high cellular toxicity. Early and effective assessment of toxicity and pharmacokinetics is essential to accelerate the drug discovery process. Conventional methods for toxicity profiling, including in vitro and in vivo assays, are laborious and resource-intensive. In response, we introduce the Small Molecule Cell Viability Database (SMCVdb), a comprehensive resource containing toxicity data for over 24 000 compounds obtained through high-content imaging (HCI). SMCVdb seamlessly integrates chemical descriptions and molecular weight data, offering researchers a holistic platform for toxicity data aiding compound prioritization and selection based on biological and economic considerations. Data collection for SMCVdb involved a systematic approach combining HCI toxicity profiling with chemical information and quality control measures ensured data accuracy and consistency. The user-friendly web interface of SMCVdb provides multiple search and filter options, allowing users to query the database based on compound name, molecular weight range, or viability percentage. SMCVdb empowers users to access toxicity profiles, molecular weights, compound names, and chemical descriptions, facilitating the exploration of relationships between compound properties and their effects on cell viability. In summary, the database provides experimentally derived cellular toxicity information for over 24 000 drug candidate molecules to academic researchers, and pharmaceutical companies. The SMCVdb will keep growing and will prove to be a pivotal resource to expedite research in drug discovery and compound evaluation. Database URL: http://smcvdb.rcb.ac.in:4321/.

CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells

BackgroundExtracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The capacity to entrap heterogeneous molecular cargoes and target specific cell populations through EV functionalization promises advancements in biomedical applications. However, the efficiency of the obtained EVs, the contribution of cell-exposed receptors to EV interactions, and the predictability of functional cargo release with potential sharing of high molecular weight recombinant mRNAs are crucial for advancing heterologous EVs in targeted therapy applications.MethodsIn this work, we selected the popular EV marker CD81 as a transmembrane guide for fusion proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. We performed high-content imaging analyses to track EV-cell interactions, including isogenic breast cancer cells with manipulated HER2 expression. We validated the functional cargo delivery of recombinant EVs carrying doxorubicin upon EV-donor cell treatment. Then, we performed an in vivo study using JIMT-1 cells commonly used as HER2-refractory, trastuzumab-resistant model to detect a more than 2000 nt length recombinant mRNA in engrafted tumors.ResultsFusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs according to their expression levels in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used to track EV-cell interactions. In time-frames where the global EV distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 exposure in isogenic cells remarkably affected the tropism of heterologous EVs, demonstrating the specificity of antiHER2 EVs representing about 20% of secreted bulk vesicles. The specific interaction strongly correlated with improved cell-killing activity of doxorubicin-EVs in MDA-MB-231 ectopically expressing HER2 and reduced toxicity in SK-BR-3 with a knocked-out HER2 receptor, overcoming the effects of the free drug. Interestingly, the fusion protein-corresponding transcripts present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, improving our sensitivity in detecting penetrant cargoes in tissue biopsies.ConclusionsThis study highlights the quantitative aspects underlying the creation of a platform for secreted heterologous EVs and shows the limits of single receptor-ligand interactions behind EV-cell engagement mechanisms, which now become the pivotal step to predict functional tropism and design new generations of EV-based nanovehicles.

Synergistic Role of Amino Acids in Enhancing mTOR Activation Through Lysosome Positioning.

Lysosome positioning, or lysosome cellular distribution, is critical for lysosomal functions in response to both extracellular and intracellular cues. Amino acids, as essential nutrients, have been shown to promote lysosome movement toward the cell periphery. Peripheral lysosomes are involved in processes such as lysosomal exocytosis, cell migration, and metabolic signaling-functions that are particularly important for cancer cell motility and growth. However, the specific types of amino acids that regulate lysosome positioning, their underlying mechanisms, and their connection to amino acid-regulated metabolic signaling remain poorly understood. In this study, we developed a high-content imaging system for unbiased, quantitative analysis of lysosome positioning. We examined the 15 amino acids present in cell culture media and found that 10 promoted lysosome redistribution toward the cell periphery to varying extents, with aromatic amino acids showing the strongest effect. This redistribution was mediated by promoting outward transport through SLC38A9-BORC-kinesin 1/3 axis and simultaneously reducing inward transport via inhibiting the recruitment of Rab7 and JIP4 onto lysosomes. When examining the effects of amino acids on mTOR activation-a central regulator of cell metabolism-we found that the amino acids most strongly promoting lysosome dispersal, such as phenylalanine, did not activate mTOR on their own. However, combining phenylalanine with arginine, which activates mTOR without affecting lysosome positioning, synergistically enhanced mTOR activity. This synergy was lost when lysosomes failed to localize to the cell periphery, as observed in kinesin 1/3 knockout (KO) cells. Furthermore, breast cancer cells exhibited heightened sensitivity to phenylalanine-induced lysosome dispersal compared to noncancerous breast cells. Inhibition of LAT1, the amino acid transporter responsible for phenylalanine uptake, reduced peripheral lysosomes and impaired cancer cell migration and proliferation, highlighting the importance of lysosome positioning in these coordinated cellular activities. In summary, amino acid-regulated lysosome positioning and mTOR signaling depend on distinct sets of amino acids. Combining lysosome-dispersing amino acids with mTOR-activating amino acids synergistically enhances mTOR activation, which may be particularly relevant in cancer cells.

Phenotypic variation in the lipopolysaccharide O-antigen of Salmonella Paratyphi A and implications for vaccine development

Enteric fever remains a major public health problem in South and Southeast Asia. The recent roll-out of the typhoid conjugate vaccine protecting against S. Typhi exhibits great promise for disease reduction in high burden areas. However, some endemic regions remain vulnerable to S. Paratyphi A due to a lack of licensed vaccines and inadequate WASH. Several developmental S. Paratyphi A vaccines exploit O-antigen as the target antigen. It has been hypothesised that O-antigen is under selective and environmental pressure, with mutations in O-antigen biosynthesis genes being reported, but their phenotypic effects are unknown. Here, we aimed to evaluate O-antigen variation in S. Paratyphi A originating from Nepal, and the potential effect of this variation on antibody binding. O-antigen variation was determined by measuring LPS laddering shift following electrophoresis; this analysis was complemented with genomic characterisation of the O-antigen region. We found structural O-antigen variation in <10 % of S. Paratyphi A organisms, but a direct underlying genetic cause could not be identified. High-content imaging was performed to determine antibody binding by commercial O2 monoclonal (mAb) and polyclonal antibodies, as well as polyclonal sera from convalescent patients naturally infected with S. Paratyphi A. Commercial mAbs detected only a fraction of an apparently “clonal” bacterial population, suggesting phase variation and nonuniform O-antigen composition. Notably, and despite visible subpopulation clusters, O-antigen structural changes did not appear to affect the binding ability of polyclonal human antibody considerably, which led to no obvious differences in the functionality of antibodies targeting organisms with different O-antigen conformations. Although these results need to be confirmed in organisms from alternative endemic areas, they are encouraging the use of O-antigen as the target antigen in S. Paratyphi A vaccines.

An adaptable, fit-for-purpose screening approach with high-throughput capability to determine speed of action and stage specificity of anti-malarial compounds.

A revamped in vitro compound identification and activity profiling approach is required to meet the large unmet need for new anti-malarial drugs to combat parasite drug resistance. Although compound hit identification utilizing high-throughput screening of large compound libraries is well established, the ability to rapidly prioritize such large numbers for further development is limited. Determining the speed of action of anti-malarial drug candidates is a vital component of malaria drug discovery, which currently occurs predominantly in lead optimization and development. This is due in part to the capacity of current methods which have low throughput due to the complexity and labor intensity of the approaches. Here, we provide an adaptable screening paradigm utilizing automated high content imaging, including the development of an automated schizont maturation assay, which collectively can identify anti-malarial compounds, classify activity into fast and slow acting, and provide an indication of the parasite stage specificity, with high-throughput capability. By frontloading these critical biological parameters much earlier in the drug discovery pipeline, it has the potential to reduce lead compound attrition rates later in the development process. The capability of the approach in its alternative formats is demonstrated using three Medicines for Malaria Venture open access compound "boxes," namely Pathogen Box (malaria set-125 compounds), Global Health Priority Box [Malaria Box 2 (80 compounds) and zoonotic neglected diseases (80 compounds)], and the Pandemic Response Box (400 compounds). From a total of 685 compounds tested, 79 were identified as having fast ring-stage-specific activity comparable to that of artemisinin and therefore of high priority for further consideration and development.

Cell-based high-content approach for SARS-CoV-2 neutralization identifies unique monoclonal antibodies and PI3K pathway inhibitors.

The sudden rise of the SARS-CoV-2 virus and the delay in the development of effective therapeutics to mitigate it made evident a need for ways to screen for compounds that can block infection and prevent further pathogenesis and spread. Yet, identifying effective drugs efficacious against viral infection and replication with minimal toxicity for the patient can be difficult. Monoclonal antibodies were shown to be effective, yet as the SARS-CoV-2 mutated, these antibodies became ineffective. Small molecule antivirals were identified using pseudovirus constructs to recapitulate infection in non-human cells, such as Vero E6 cells. However, the impact was limited due to poor translation of these compounds in the clinical setting. This is partly due to the lack of similarity of screening platforms to the in vivo physiology of the patient and partly because drugs effective in vitro showed dose-limiting toxicities. In this study, we performed two high-throughput screens in human lung adenocarcinoma cells with authentic SARS-CoV-2 virus to identify both monoclonal antibodies that neutralize the virus and clinically useful kinase inhibitors to block the virus and prioritize minimal host toxicity. Using high-content imaging combined with single-cell and multidimensional analysis, we identified antibodies and kinase inhibitors that reduce virus infection without affecting the host. Our screening technique uncovered novel antibodies and overlooked kinase inhibitors (i.e. PIK3i, mTORi, multiple RTKi) that could be effective against SARS-CoV-2 virus. Further characterization of these molecules will streamline the repurposing of compounds for the treatment of future pandemics and uncover novel mechanisms viruses use to hijack and infect host cells.

Scalable, compressed phenotypic screening using pooled perturbations.

High-throughput phenotypic screens using biochemical perturbations and high-content readouts are constrained by limitations of scale. To address this, we establish a method of pooling exogenous perturbations followed by computational deconvolution to reduce required sample size, labor and cost. We demonstrate the increased efficiency of compressed experimental designs compared to conventional approaches through benchmarking with a bioactive small-molecule library and a high-content imaging readout. We then apply compressed screening in two biological discovery campaigns. In the first, we use early-passage pancreatic cancer organoids to map transcriptional responses to a library of recombinant tumor microenvironment protein ligands, uncovering reproducible phenotypic shifts induced by specific ligands distinct from canonical reference signatures and correlated with clinical outcome. In the second, we identify the pleotropic modulatory effects of a chemical compound library with known mechanisms of action on primary human peripheral blood mononuclear cell immune responses. In sum, our approach empowers phenotypic screens with information-rich readouts to advance drug discovery efforts and basic biological inquiry.

Enhancing quantitative imaging to study DNA damage response: A guide to automated liquid handling and imaging

Laboratory automation and quantitative high-content imaging are pivotal in advancing diverse scientific fields. These innovative techniques alleviate the burden of manual labour, facilitating large-scale experiments characterized by exceptional reproducibility. Nonetheless, the seamless integration of such systems continues to pose a constant challenge in many laboratories. Here, we present a meticulously designed workflow that automates the immunofluorescence staining process, coupled with quantitative high-content imaging to study DNA damage signalling as an example. This is achieved by using an automatic liquid handling system for sample preparation. Additionally, we also offer practical recommendations aimed at ensuring the reproducibility and scalability of experimental outcomes. We illustrate the high level of efficiency and reproducibility achieved through the implementation of the liquid handling system but also addresses the associated challenges. Furthermore, we extend the discussion into critical aspects such as microscope selection, optimal objective choices, and considerations for high-content image acquisition. Our study streamlines the image analysis process, offering valuable recommendations for efficient computing resources and the integration of cutting-edge deep learning techniques. Emphasizing the paramount importance of robust data management systems aligned with the FAIR data principles, we provide practical insights into suitable storage options and effective data visualization techniques. Together, our work serves as a comprehensive guide for life science laboratories seeking to elevate their high-content quantitative imaging capabilities through the seamless integration of advanced laboratory automation.

Investigating the impact of botulinum toxin type a on the migration of normal human dermal fibroblasts: An invitro wound healing assay.

Botulinum toxin A (BoNT-A) is widely utilized in the management of hypertrophic and keloid scars. One proposed mechanism for scar prevention involves the inhibition of fibroblast migration in scars by BoNT-A. However, the data regarding the effect of BoNT-A on the migration of normal human dermal fibroblasts (NHDF) is limited. The aim of this study was to investigate the inhibitory effect of different types and dilutions of BoNT-A on the migration of NHDF. In vitro scratch wound assay, NHDF cells were cultured, incubated, and subjected to scratching using a sterile tip. Subsequently, the scratched NHDF monolayer was treated with different types of BoNT-A, including onabotulinumtoxinA (ONA), incobotulinumtoxinA (INCO), prabotulinumtoxinA (PRABO), or letibotulinumtoxinA (LETI), at varying concentrations of 10, 20, 25, 40, 50, and 100 units/milliliter (U/mL). Additionally, abobotulinumtoxinA (ABO) was administered at concentrations of 33, 50, 66, 71, 100, 150, 300, and 500 U/mL. Normal saline solution (NSS) served as a negative control. The extent of NHDF migration was evaluated by comparing each dilution of BoNT-A with the controls using high-content imaging at the 48-h time point. Furthermore, the viability of the of NHDF was assessed. The concentrations of 25, 40, and 50 U/mL of ONA (p < 0.001) and 25 U/mL of LETI (p < 0.05) demonstrated significantly inhibited NHDF migration in comparison to the control group. Conversely, all dilutions of PRABO, INCO, and ABO exhibited comparable NHDF migration to that of the control group. Regarding NHDF viability, no significant decrease was observed across any of the BoNT-A types and dilutions. Different types and dilutions of BoNT-A demonstrated variable inhibitory effects on NHDF migration invitro. The selection of BoNT-A formulation may significantly impact the clinical outcome of scar prevention related to fibroblast migration.

A High-Content and High-Throughput Imaging Screen to Identify a Small Molecule Inhibitor for the Lipid Uptake Function of KIM-1

A High-Content and High-Throughput Imaging Screen to Identify a Small Molecule Inhibitor for the Lipid Uptake Function of KIM-1

224 (PB212): AI-enhanced high-content imaging to identify effective compound combinations with CDK4/6 inhibitor and endocrine therapy in ER+ HER2- breast cancer

224 (PB212): AI-enhanced high-content imaging to identify effective compound combinations with CDK4/6 inhibitor and endocrine therapy in ER+ HER2- breast cancer

Rapid full-color serial sectioning tomography with speckle illumination and ultraviolet excitation

Three-dimensional (3D) high-resolution large-volume imaging has remained a challenge. Translational rapid ultraviolet-excited sectioning tomography (TRUST) achieves rapid and cost-effective whole-organ subcellular imaging through iterative optical scanning and mechanical sectioning. However, the axial resolution is limited by the mechanical sectioning thickness or the UV light penetration depth in tissue. Here, assisted with high-and-low-frequency (HiLo) microscopy (HiLoTRUST), the optical sectioning thickness has been reduced from tens of micrometers to ~5.8 µm. In addition, HiLoTRUST has attained a finer mechanical sectioning thickness (10–15 µm) compared to TRUST (50 µm). For high-content imaging as in TRUST, we employed two additional UV light-emitting diodes (LEDs) specifically for uniform illumination. The full-color imaging ability and improved axial resolution of HiLoTRUST have been validated by two-dimensional (2D)/3D imaging of various mouse organs and human lung cancer specimens. HiLoTRUST offers a cost-effective, full-color, and high-resolution 3D imaging approach, showing its great potential in 3D histology applications.

Dually Labeled Neurotensin NTS1R Ligands for Probing Radiochemical and Fluorescence-Based Binding Assays.

The determination of ligand-receptor binding affinities plays a key role in the development process of pharmaceuticals. While the classical radiochemical binding assay uses radioligands, fluorescence-based binding assays require fluorescent probes. Usually, radio- and fluorescence-labeled ligands are dissimilar in terms of structure and bioactivity, and can be used in either radiochemical or fluorescence-based assays. Aiming for a close comparison of both assay types, we synthesized tritiated fluorescent neurotensin receptor ligands ([3H]13, [3H]18) and their nontritiated analogues (13, 18). The labeled probes were studied in radiochemical and fluorescence-based (high-content imaging, flow cytometry, fluorescence anisotropy) binding assays. Equilibrium saturation binding yielded well-comparable ligand-receptor affinities, indicating that all these setups can be used for the screening of new drugs. In contrast, discrepancies were found in the kinetic behavior of the probes, which can be attributed to technical differences of the methods and require further studies with respect to the elucidation of the underlying mechanisms.

High-throughput drug screening identifies novel therapeutics for Low Grade Serous Ovarian Carcinoma

Low grade serous carcinoma (LGSOC) is a rare epithelial ovarian cancer with unique molecular characteristics compared to the more common tubo-ovarian high-grade serous ovarian carcinoma. Pivotal clinical trials guiding the management of epithelial ovarian cancer lack sufficient cases of LGSOC for meaningful subgroup analysis, hence overall findings cannot be extrapolated to rarer chemo-resistant subtypes such as LGSOC. Furthermore, there is a need for more effective therapies for the treatment of relapsed disease, as treatment options are limited. To address this, we conducted the largest quantitative high-throughput drug screening effort (n = 3436 compounds) in 12 patient-derived LGSOC cell lines and one normal ovary cell line to identify unexplored therapeutic avenues. Using a combination of high-throughput robotics, high-content imaging and novel data analysis pipelines, our data set identified 60 high and 19 moderate confidence hits which induced cancer cell specific cytotoxicity at the lowest compound dose assessed (0.1 µM). We also revealed a series of known (mTOR/PI3K/AKT) and novel (EGFR and MDM2-p53) drug classes in which LGSOC cell lines showed demonstrable susceptibility to.

Mid-infrared chemical imaging of living cells enabled by plasmonic metasurfaces.

Mid-Infrared (MIR) chemical imaging provides rich chemical information of biological samples in a label-free and non-destructive manner. Yet, its adoption to live-cell analysis is limited by the strong attenuation of MIR light in water, often necessitating cell culture geometries that are incompatible with the prolonged viability of cells and with standard high-throughput workflow. Here, we introduce a new approach to MIR microscopy, where cells are imaged through their localized near-field interaction with a plasmonic metasurface. Chemical contrast of distinct molecular groups provided sub-cellular resolution images of the proteins, lipids, and nucleic acids in the cells that were collected using an inverted MIR microscope. Time-lapse imaging of living cells demonstrated that their behaviors, including motility, viability, and substrate adhesion, can be monitored over extended periods of time using low-power MIR light. The presented approach provides a method for the non-perturbative MIR imaging of living cells, which is well-suited for integration with modern high-throughput screening technologies for the label-free, high-content chemical imaging of living cells.

Comparative Analysis of Canonical Inflammasome Activation by Flow Cytometry, Imaging Flow Cytometry and High-Content Imaging.

Inflammasome activation occurs in various diseases, including rare diseases that require multicenter studies for investigation. Flow cytometric analysis of ASC speck+ cells in patient samples can be used to detect cell type-specific inflammasome activation. However, this requires standardized sample processing and the ability to compare data from different flow cytometers. To address this issue, we analyzed stimulated and unstimulated PBMCs from healthy donors using seven different flow cytometers. Additionally, human PBMCs were analyzed by fluorescence microscopy, imaging flow cytometry and high-content imaging (HCI). Flow cytometers differed significantly in their ability to detect ASC speck+ cells. AriaIII, Astrios EQ, and Canto II performed best in separating ASC speck+ from diffuse ASC cells. Imaging flow cytometry and HCI provided additional insight into ASC speck formation based on image-based parameters. For optimal results, the ability to separate cells with diffuse ASC from ASC speck+ cells is decisive. Image-based parameters can also differentiate cells with diffuse ASC from ASC speck+ cells. For the first time, we analyzed ASC speck detection by HCI in PBMCs and demonstrated advantages of this technique, such as high-throughput, algorithm-driven image quantification and 3D-rendering. Thus, inflammasome activation by ASC speck formation can be detected by various technical methods. However, the results may vary depending on the device used.

A high content imaging assay for identification of specific inhibitors of native Plasmodium liver stage protein synthesis.

Plasmodium parasite resistance to antimalarial drugs is a serious threat to public health in malaria-endemic areas. Compounds that target core cellular processes like translation are highly desirable, as they should be capable of killing parasites in their liver and blood stage forms, regardless of molecular target or mechanism. Assays that can identify these compounds are thus needed. Recently, specific quantification of native Plasmodium berghei liver stage protein synthesis, as well as that of the hepatoma cells supporting parasite growth, was achieved via automated confocal feedback microscopy of the o-propargyl puromycin (OPP)-labeled nascent proteome, but this imaging modality is limited in throughput. Here, we developed and validated a miniaturized high content imaging (HCI) version of the OPP assay that increases throughput, before deploying this approach to screen the Pathogen Box. We identified only two hits; both of which are parasite-specific quinoline-4-carboxamides, and analogs of the clinical candidate and known inhibitor of blood and liver stage protein synthesis, DDD107498/cabamiquine. We further show that these compounds have strikingly distinct relationships between their antiplasmodial and translation inhibition efficacies. These results demonstrate the utility and reliability of the P. berghei liver stage OPP HCI assay for the specific, single-well quantification of Plasmodium and human protein synthesis in the native cellular context, allowing the identification of selective Plasmodium translation inhibitors with the highest potential for multistage activity.

Multiplexed High Content Imaging assay optimization for the analysis of apoptosis/necrosis in breast cancer cells after treatment with Vorinostat analogues

Multiplexed High Content Imaging assay optimization for the analysis of apoptosis/necrosis in breast cancer cells after treatment with Vorinostat analogues

High Content Screening Assay of Inhibitors of the Legionellapneumophila Histone Methyltransferase RomA in Infected Cells.

Resistance to anti-microbial agents is a world-wide health threat. Thus there is an urgent need for new treatments. An alternative approach to disarm pathogens consists in developing drugs targeting epigenetic modifiers. Bacterial pathogens can manipulate epigenetic regulatory systems of the host to bypass defences to proliferate and survive. One example is Legionella pneumophila, a Gram-negative intracellular pathogen that targets host chromatin with a specific, secreted bacterial SET-domain methyltransferase named RomA. This histone methyltransferase specifically methylates H3K14 during infection and is responsible for changing the host epigenetic landscape upon L. pneumophila infection. To inhibit RomA activity during infection, we developed a reliable high-content imaging screening assay, which we used to screen an in-house chemical library developed to inhibit DNA and histone methyltransferases. This assay was optimised using monocytic leukemic THP-1 cells differentiated into macrophages infected with L. pneumophila in a 96- or 384-well plate format using the Opera Phenix® (Perkin Elmer) confocal microscope, combined with Columbus™ software for automated image acquisition and analysis. H3K14 methylation was followed in infected, single cells and cytotoxicity was assessed in parallel. A first pilot screening of 477 compounds identified a potential starting point for inhibitors of H3K14 methylation.