Isolation Of Nuclei Research Articles

Optimization of ATAC-seq in wheat seedling roots using INTACT-isolated nuclei

BackgroundThe genetic information contained in the genome of an organism is organized in genes and regulatory elements that control gene expression. The genomes of multiple plants species have already been sequenced and the gene repertory have been annotated, however, cis-regulatory elements remain less characterized, limiting our understanding of genome functionality. These elements act as open platforms for recruiting both positive- and negative-acting transcription factors, and as such, chromatin accessibility is an important signature for their identification.ResultsIn this work we developed a transgenic INTACT [isolation of nuclei tagged in specific cell types] system in tetraploid wheat for nuclei purifications. Then, we combined the INTACT system together with the assay for transposase-accessible chromatin with sequencing [ATAC-seq] to identify open chromatin regions in wheat root tip samples. Our ATAC-seq results showed a large enrichment of open chromatin regions in intergenic and promoter regions, which is expected for regulatory elements and that is similar to ATAC-seq results obtained in other plant species. In addition, root ATAC-seq peaks showed a significant overlap with a previously published ATAC-seq data from wheat leaf protoplast, indicating a high reproducibility between the two experiments and a large overlap between open chromatin regions in root and leaf tissues. Importantly, we observed overlap between ATAC-seq peaks and cis-regulatory elements that have been functionally validated in wheat, and a good correlation between normalized accessibility and gene expression levels.ConclusionsWe have developed and validated an INTACT system in tetraploid wheat that allows rapid and high-quality nuclei purification from root tips. Those nuclei were successfully used to performed ATAC-seq experiments that revealed open chromatin regions in the wheat genome that will be useful to identify cis-regulatory elements. The INTACT system presented here will facilitate the development of ATAC-seq datasets in other tissues, growth stages, and under different growing conditions to generate a more complete landscape of the accessible DNA regions in the wheat genome.

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Resolving the thiazide-induced hypocalciuria conundrum using single cell transcriptomics

Thiazide diuretics lower urinary calcium excretion and are used to prevent calcium stone recurrence. Thiazides inhibit the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Hypocalciuria also occurs in Gitelman syndrome, caused by mutations in SLC12A3, encoding NCC. The mechanism by which loss of NCC function affects calcium handling remains very controversial. The two prevailing hypotheses invoked are 1) hypocalciuria results from volume-dependent increases in proximal tubule reabsorption; 2) thiazides activate calcium reabsorption along the DCT. Here we combined single nucleus RNA sequencing with physiologic analysis to unravel the causes of hypocalciuria.Eight to 10-week old male mice were given 50 mg/kg/day metolazone (MTZ) orally in food for 4 days. Littermates receiving only vehicle were used as controls. As DCT cells comprise a small percentage of the kidney mass, we used the NCC-Cre +/- - INTACT +/- mouse line to enrich for DCT via Cre-dependent GFP expression in DCT nuclei. After nuclei isolation, GFP+ events (DCT nuclei) were enriched by fluorescence assisted nuclei sorting. The purified DCT nuclei were sequenced using 10X Chromium controller. Reads were then analyzed using a standard transcriptomic bioinformatics pipeline with Seurat.The MTZ-treated mice showed lower urinary calcium excretion compared to controls recapitulating thiazide-induced hypocalciuria. Our snRNAseq integrated dataset contained 51,876 mapped genes across 45,953 samples from 3 MTZ-treated and 3 control mice. Roughly 99% of the nuclei were singlets expressing Slc12a3 showing successful enrichment for the DCT. Dimensional reduction analysis and projection confirmed two clusters, DCT1 and DCT2, based on canonical segment markers consistent with previously published transcriptomic atlases. Gene expression analysis showed the DCT2 contained a calcium cassette consisting of the known calciotropic genes Trpv5, Calb1, S100g, Slc8a1, Ryr2, Vdr, Casr, and Atp2b1. These genes were also expressed in the connecting tubule (CNT), as documented using the Calb1-Cre-INTACT system. From this set of genes, we defined a calcium score computed from the pooled average expression of all these genes in a single cell. Although all calcium cassette genes, and the average calcium score, were lower in the MTZ-treated group, the proportion of DCT2/DCT1 cells increased (0.27 ± 0.03 vs 0.35 ± 0.03; p = 0.027). These data suggest that lower calcium delivery to the DCT reduces calcium cassette expression per cell, but that distal fractional reabsorption is nonetheless increased owing to hypertrophy and elongation of the calcium transporting segments, DCT2 and CNT.We propose a modified mechanism to account for thiazide -induced hypocalciuria: 1) volume-dependent increase in proximal tubule reabsorption; 2) increased fractional (not absolute) DCT2 reabsorption owing to structural remodeling of DCT2 and CNT providing more surface area for calcium reabsorption. Funding: NIH DK51496, NIH DK054983, VA 1I01BX002228, LeDucq Foundation, DOST-PCHRD-ASTHRDP This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Analysis of distal convoluted tubule (DCT) cell de-differentiation due to COP9 signalosome (CSN) dysfunction via enriched DCT single-nucleus RNA-sequencing

Cullin-RING-ligases (CRLs) are a group of E3 ubiquitin ligases that mediate the regulated degradation of proteins. All CRLs are regulated by the COP9 signalosome (CSN), which interacts with the CRL and turns off ubiquitin ligase activity. To investigate the role of impaired CSN dysfunction in the kidney, we inactivated the CSN by deleting Jab1 (the key CSN catalytic subunit) along the entire nephron. Even though the CSN is expressed all along the nephron, these mice showed remodeling only along the distal nephron, with shortening of the distal convoluted tubule (DCT) and a large reduction in DCT-specific proteins. The DCT is known to remodel due to changes in dietary salt intaked, drug administration, and human disease. Yet, the cellular and molecular basis for this plasticity is unclear. We hypothesized that CSN dysfunction causes de-differentiation of DCT cells leading to DCT remodeling. To test our hypothesis, inducible DCT-specific NCC-Cre-ERT2 mice were bred to INTACT (Isolation of Nuclei TAgged in specific Cell Types, which fluorescently labels nuclei) and Jab1 floxed mice to generate DCT-INTACT- Jab1 -/- mice. One adult male DCT-INTACT- Jab1 -/- mouse and one DCT-INTACT control mouse were treated with tamoxifen to induce nuclear GFP expression and to induce deletion of Jab1 only in DCT cells. Six-weeks post treatment, nuclei from one kidney cortex was isolated and the DCT was enriched using Fluorescence-Activated Nuclei Sorting (FANS). GFP-positive nuclei were used for single-nucleus RNA-sequencing (NovaSeq). Initial results showed that unbiased clustering and UMAP visualization revealed 7 clusters with most cells from the DCT (99.0% for control and 97.6% for Jab1 -/- mice), indicating that the enrichment process was highly successful. Both the control and Jab1 -/- mouse had similar proportions of DCT1 and DCT2 cells; however, we identified two specialized cell types (dDCT1 and dDCT2) which comprised a much larger proportion of the cells in the Jab1 -/- mouse (dDCT1: 2.9% in control vs 41.1% in Jab1 -/- ; dDCT2: 2.0% in control vs 13.9% in Jab1 -/- ). These cells were highly de-differentiated with lower expression of the canonical DCT identifier genes such as Egf and Umod for DCT1, Calb1 and S100g for DCT2, as well as Slc12a3. Both dDCT1 and dDCT2 cell populations had induced expression of the disease- or carcinoma-related genes Ano3, Nrg1, and Pvt1. In conclusion, Jab1 deletion causes de-differentiation of a population of DCT cells. Further examination of CSN dysfunction via DCT-enriched single-nucleus RNA-sequencing could lead to a better understanding of the molecular mechanisms involved in DCT remodeling. NIH K01DK120790, NIH R01DK51496, Foundation LeDucq: Transatlantic Network of Excellence 17CVD05 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modified CTAB protocols for high-molecular-weight DNA extractions from ferns.

Efficient protocols for extracting high-molecular-weight (HMW) DNA from ferns facilitate the long-read sequencing of their large and complex genomes. Here, we perform two cetyltrimethylammonium bromide (CTAB)-based protocols to extract HMW DNA and evaluate their applicability in diverse fern taxa for the first time. We describe two modified CTAB protocols, with key adjustments to minimize mechanical disruption during lysis to prevent DNA shearing. One of these protocols uses a small amount of fresh tissue but yields a considerable quantity of HMW DNA with high efficiency. The other accommodates a large amount of input tissue, adopts an initial step of nuclei isolation, and thus ensures a high yield in a short period of time. Both methods were proven to be robust and effective in obtaining HMW DNA from diverse fern lineages, including 33species in 19 families. The DNA extractions mostly had high DNA integrity, with mean sizes larger than 50 kbp, as well as high purity (A260/A230 and A260/A280 > 1.8). This study provides HMW DNA extraction protocols for ferns in the hope of facilitating further attempts to sequence their genomes, which will bridge our genomic understanding of land plant diversity.

In situ isolation of nuclei or nuclear proteins from adherent cells: a simple, effective method with less cytoplasmic contamination

BackgroundIsolation of nuclei or nuclear proteins is a prerequisite for western blot, nuclear proteome profiling, and other evaluations of nuclear proteins. Here, we developed a simple method for in situ isolation of nuclei or nuclear proteins by in situ removing the extranuclear part of adherent cells via a classical nonionic detergent triton X-100.ResultsFirst, the feasibility of our method was confirmed by confocal microscopy, atomic force microscopy, scanning electron microscopy, dynamic light scattering, immunofluorescence imaging, and time-lapse dynamic observation. Next, the optimal concentration range (approximately 0.1–1% for ~ 10 min) of triton X-100 and the optimal treatment time (< 30 min) of 0.1–1% Triton X-100 for our method were determined via western blotting of eight extra-/intra-nuclear proteins. Subsequently, the effectiveness, sensitivity, and cytoplasmic contamination of our method were tested by investigating the levels of phosphorylated p65 (a NF-κB subunit) in the nuclei of endothelial or tumor cells treated with/without lipopolysaccharide (LPS) via western blotting and by comparing with a commercial nuclear protein extraction kit (a classical detergent-based method). The data show that compared with the commercial kit our method obtained a higher yield of total nuclear proteins, a higher pP65 level in both control and LPS groups, and much lower content of GAPDH (as a reference for cytoplasmic contamination) in nuclei.ConclusionsThe in situ isolation of nuclei or nuclear proteins from adherent cells in this study is a simple, effective method with less cytoplasmic contamination. This method/strategy has the potential of improving the quality of downstream evaluations including western blotting and proteomic profiling.

High-quality nuclei isolation from postmortem human heart muscle tissues for single-cell studies

Single-cell approaches have become an increasingly popular way of understanding the genetic factors behind disease. Isolation of DNA and RNA from human tissues is necessary to analyze multi-omic data sets, providing information on the single-cell genome, transcriptome, and epigenome. Here, we isolated high-quality single-nuclei from postmortem human heart tissues for DNA and RNA analysis. Postmortem human tissues were obtained from 106 individuals, 33 with a history of myocardial disease, diabetes, or smoking, and 73 controls without heart disease. We demonstrated that the Qiagen EZ1 instrument and kit consistently isolated genomic DNA of high yield, which can be used for checking DNA quality before conducting single-cell experiments. Here, we provide a method for single-nuclei isolation from cardiac tissue, otherwise known as the SoNIC method, which allows for the isolation of single cardiomyocyte nuclei from postmortem tissue by nuclear ploidy status. We also provide a detailed quality control measure for single-nuclei whole genome amplification and a pre-amplification method for confirming genomic integrity.

Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies

ABSTRACT This paper provides a laboratory workflow for single-nucleus RNA-sequencing (snRNA-seq) including a protocol for gentle nuclei isolation from fresh frozen tumor biopsies, making it possible to analyze biobanked material. To develop this protocol, we used non-frozen and frozen human bladder tumors and cell lines. We tested different lysis buffers (IgePal and Nuclei EZ) and incubation times in combination with different approaches for tissue and cell dissection: sectioning, semi-automated dissociation, manual dissociation with pestles, and semi-automated dissociation combined with manual dissociation with pestles. Our results showed that a combination of IgePal lysis buffer, tissue dissection by sectioning, and short incubation time was the best conditions for gentle nuclei isolation applicable for snRNA-seq, and we found limited confounding transcriptomic changes based on the isolation procedure. This protocol makes it possible to analyze biobanked material from patients with well-described clinical and histopathological information and known clinical outcomes with snRNA-seq.

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome.

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

Chromatin Immunoprecipitation (ChIP) to Study the Transcriptional Regulatory Network that Controls Iron Homeostasis in Arabidopsis thaliana.

In plants, gene expression is orchestrated by thousands of transcription factors (TFs). For instance, a large set of bHLH TFs are involved in the regulation of iron homeostasis in Arabidopsis thaliana. The identification of the direct target genes of TFs through uncovering the interaction between the TFs and cis-regulatory elements has become an essential step toward a comprehensive understanding of the iron homeostasis transcriptional regulatory network in Arabidopsis. Chromatin immunoprecipitation (ChIP) followed by qRT-PCR (ChIP-qPCR), sequencing (ChIP-seq), or chip hybridization (ChIP-chip) is a robust tool to investigate protein-DNA interactions in plants in a physiological context. The procedure generally includes six steps: DNA-protein crosslink, isolation of nuclei, shearing of chromatin, immunoprecipitation, DNA purification, and qRT-PCR analyses. In this protocol, we describe guidelines, experimental setup, and conditions for ChIP experiment in Arabidopsis. This protocol focuses on seedlings grown in control and iron deficiency conditions, but can readily be adapted for use with other Arabidopsis tissues or samples. In addition, the protocol could also be applied to perform ChIP-chip or ChIP-seq experiments.

Isolation of Nuclei from Human Snap-frozen Liver Tissue for Single-nucleus RNA Sequencing.

Single-nucleus RNA sequencing (snRNA-seq) provides a powerful tool for studying cell type composition in heterogenous tissues. The liver is a vital organ composed of a diverse set of cell types; thus, single-cell technologies could greatly facilitate the deconvolution of liver tissue composition and various downstream omics analyses at the cell-type level. Applying single-cell technologies to fresh liver biopsies can, however, be very challenging, and snRNA-seq of snap-frozen liver biopsies requires some optimization given the high nucleic acid content of the solid liver tissue. Therefore, an optimized protocol for snRNA-seq specifically targeted for the use of frozen liver samples is needed to improve our understanding of human liver gene expression at the cell-type resolution. We present a protocol for performing nuclei isolation from snap-frozen liver tissues, as well as guidance on the application of snRNA-seq. We also provide guidance on optimizing the protocol to different tissue and sample types.

Methods for Single Cell Transcriptomic Analysis of Adipose Tissue.

The development of single cell approaches has facilitated the investigation of cellular heterogeneity and cell type-specific gene expression in complex tissues. Adipose tissue depots contain lipid storing adipocytes as well as a diverse array of cell types that form the adipocyte niche and regulate adipose tissue function. Here, I describe two protocols for the isolation of single cells and nuclei from white and brown adipose tissue. Additionally, I provide a detailed workflow for isolation of cell type- or lineage-specific single nuclei using nuclear tagging and translating ribosome affinity purification (NuTRAP) mouse models.

Isolation of Nuclei Tagged in Specific Cell Types (INTACT) in Arabidopsis.

Many functionally distinct plant tissues have relatively low numbers of cells that are embedded within complex tissues. For example, the shoot apical meristem (SAM) consists of a small population of pluripotent stem cells surrounded by developing leaves and/or flowers at the growing tip of the plant. It is technically challenging to collect enough high-quality SAM samples for molecular analyses. Isolation of Nuclei Tagged in specific Cell Types (INTACT) is an easily reproducible method that allows the enrichment of biotin-tagged cell-type-specific nuclei from the total nuclei pool using biotin-streptavidin affinity purification. Here, we provide a detailed INTACT protocol for isolating nuclei from the Arabidopsis SAM. One can also adapt this protocol to isolate nuclei from other tissues and cell types for investigating tissue/cell-type-specific transcriptome and epigenome and their changes during developmental programs at a high spatiotemporal resolution. Furthermore, due to its low cost and simple procedures, INTACT can be conducted in any standard molecular laboratory.

Plant Nuclei Isolation for Single-Nucleus RNA Sequencing.

Transcriptome profiling has been significantly hampered by the heterogeneity among individual cells within a tissue or an organ. Recent advances in single cell transcriptome profiling have significantly advanced our understanding of the transcriptome. However, plant single-cell RNA sequencing (scRNA-seq) relies on the isolation of protoplasts, which is not only impossible for many cell types but also induces acute wounding responses. To solve these problems, single-nucleus RNA sequencing (snRNA-seq) has been applied to plant research, in which nuclei are isolated and subject to encapsulation and profiling. Compared with scRNA-seq, snRNA-seq can be applied to a wider range of tissue types and plant species. Nevertheless, fewer transcripts can be obtained from each nucleus than each protoplast. In this chapter, we describe a detailed and general protocol to prepare nuclei from plant tissues that are ready for subsequent library construction and high-throughput sequencing.

The eINTACT system dissects bacterial exploitation of plant osmosignalling to enhance virulence

Bacteria inject effector proteins into host cells to manipulate cellular processes that promote disease. Since bacteria deliver minuscule amounts of effectors only into targeted host cells, it is technically challenging to capture effector-dependent cellular changes from bulk-infected host tissues. Here, we report a new technique called effector-inducible isolation of nuclei tagged in specific cell types (eINTACT), which facilitates affinity-based purification of nuclei from Arabidopsis plant cells that have received Xanthomonas bacterial effectors. Analysis of purified nuclei reveals that the Xanthomonas effector XopD manipulates the expression of Arabidopsis abscisic acid signalling-related genes and activates OSCA1.1, a gene encoding a calcium-permeable channel required for stomatal closure in response to osmotic stress. The loss of OSCA1.1 causes leaf wilting and reduced bacterial growth in infected leaves, suggesting that OSCA1.1 promotes host susceptibility. eINTACT allows us to uncover that XopD exploits host OSCA1.1/abscisic acid osmosignalling-mediated stomatal closure to create a humid habitat that favours bacterial growth and opens up a new avenue for accurately elucidating functions of effectors from numerous gram-negative plant bacteria in native infection contexts.

Cell-type specific profiling of histone post-translational modifications in the adult mouse striatum

Epigenetic gene regulation in the heterogeneous brain remains challenging to decipher with current strategies. Bulk tissue analysis from pooled subjects reflects the average of cell-type specific changes across cell-types and individuals, which obscures causal relationships between epigenetic modifications, regulation of gene expression, and complex pathology. To address these limitations, we optimized a hybrid protocol, ICuRuS, for the isolation of nuclei tagged in specific cell-types and histone post translational modification profiling from the striatum of a single mouse. We combined affinity-based isolation of the medium spiny neuron subtypes, Adenosine 2a Receptor or Dopamine Receptor D1, with cleavage of histone-DNA complexes using an antibody-targeted micrococcal nuclease to release DNA complexes for paired end sequencing. Unlike fluorescence activated cell sorting paired with chromatin immunoprecipitation, ICuRuS allowed for robust epigenetic profiling at cell-type specific resolution. Our analysis provides a framework to understand combinatorial relationships between neuronal-subtype-specific epigenetic modifications and gene expression.

Isolation of Nuclei from Flash-frozen Liver Tissue for Single-cell Multiomics.

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease. This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Isolation of Nuclei from Flash-frozen Liver Tissue for Single-cell Multiomics

Isolation Nuclei From Flash Frozen Liver Tissue For Single Cell - Video. JoVE publishes peer-reviewed scientific video protocols to accelerate biological, medical, chemical and physical research. Watch our scientific video articles. JoVE is the world-leading producer and provider of science videos with the mission to improve scientific research, scientific journals, and education. Millions of scientists, educators and students at thousands of universities, colleges, hospitals and biopharmaceutical companies worldwide use JoVE for their research, teaching and learning.

Systematic Methods for Isolating High Purity Nuclei from Ten Important Plants for Omics Interrogation

Recent advances in developmental biology have been made possible by using multi-omic studies at single cell resolution. However, progress in plants has been slowed, owing to the tremendous difficulty in protoplast isolation from most plant tissues and/or oversize protoplasts during flow cytometry purification. Surprisingly, rapid innovations in nucleus research have shed light on plant studies in single cell resolution, which necessitates high quality and efficient nucleus isolation. Herein, we present efficient nuclei isolation protocols from the leaves of ten important plants including Arabidopsis, rice, maize, tomato, soybean, banana, grape, citrus, apple, and litchi. We provide a detailed procedure for nucleus isolation, flow cytometry purification, and absolute nucleus number quantification. The nucleus isolation buffer formula of the ten plants tested was optimized, and the results indicated a high nuclei yield. Microscope observations revealed high purity after flow cytometry sorting, and the DNA and RNA quality extract from isolated nuclei were monitored by using the nuclei in cell division cycle and single nucleus RNA sequencing (snRNA-seq) studies, with detailed procedures provided. The findings indicated that nucleus yield and quality meet the requirements of snRNA-seq, cell division cycle, and likely other omic studies. The protocol outlined here makes it feasible to perform plant omic studies at single cell resolution.

The Osteosarcoma Stem Cell Activity of a Gallium(III)-Phenanthroline Complex Appended to Salicylate.

We report the synthesis, characterisation, and anti-osteosarcoma properties of a gallium(III) complex (1) comprising of two 1,10-phenanthroline ligands and salicylate, a non-steroidal anti-inflammatory drug. The gallium(III) complex 1 displays micromolar potency towards bulk osteosarcoma cells and osteosarcoma stem cells (OSCs). Notably, the gallium(III) complex 1 exhibits significantly higher toxicity towards OSCs grown in monolayer and three-dimensional cultures than cisplatin, a frontline anti-osteosarcoma drug. Nuclei isolation and immunoblotting studies show that the gallium(III) complex 1 enters osteosarcoma cell nuclei and induces DNA damage. Flow cytometry and cytotoxicity studies (in the presence of prostaglandin E2) indicate that the gallium(III) complex 1 downregulates cyclooxygenase-2 (COX-2) expression and kills osteosarcoma cells in a COX-2-dependent manner. Further, the mode of osteosarcoma cell death evoked by the gallium(III) complex 1 is characterised as caspase-dependent apoptosis.