Cell-permeable Probe Research Articles

Abstract A005: A live cell PRMT5 NanoBRET™ target engagement assay querying competitive and uncompetitive modes of inhibition

Abstract PRMT5 is an essential arginine methyltransferase that regulates a wide spectrum of cellular processes through the methylation of histone and non-histone substrates. In PRMT5 catalysis, SAM serves as the cofactor and methyl group donor, generating a methylated guanidinium moiety on the target substrate. In normal cells, the SAM pools are maintained through the methionine salvage pathway. In 15% of cancers, a key enzyme in this pathway (MTAP) is deleted, leading to an accumulation of the intermediate MTA, which inhibits PRMT5 activity. This genetic loss of function has therefore been pursued as a collateral vulnerability in MTAP deleted cancers. To exploit this synthetic lethality, a number of novel inhibitors (such as MRTX1719) have been developed that bind cooperatively at the PRMT5/MTA complex, offering a compelling pathway to precision medicine. Here we describe a novel, live cell NanoBRET™ Target Engagement assay that enables mechanistic studies on a variety of PRMT5 inhibitors. Using a cell-permeable NanoBRET probe directed to the substrate pocket of PRMT5, both substrate- and cofactor-competitive engagement can be quantified in cells. Moreover, this method can be used to quantify MTA-uncompetitive target engagement in cells, providing a platform to measure the potency of PRMT5-MTA-drug ternary complex formation. This method serves as a first-in-class method to quantify uncompetitive target engagement in live cells, which can be applied to other model systems. Citation Format: Kelly A. Teske, Ani Michaud, Elisabeth Rothweiler, Cesear Corona, Kaitlin Dunn Hoffman, Jennifer Wilkinson, Michael Beck, James Vasta, Kilian Huber, Matthew Robers. A live cell PRMT5 NanoBRET™ target engagement assay querying competitive and uncompetitive modes of inhibition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A005.

Enabling Universal Access to Rapid and Stable Tetrazine Bioorthogonal Probes through Triazolyl-Tetrazine Formation.

Despite the immense potential of tetrazine bioorthogonal chemistry in biomedical research, the in vivo performance of tetrazine probes is challenged by the inverse correlation between the physiological stability and reactivity of tetrazines. Additionally, the synthesis of functionalized tetrazines is often complex and requires specialized reagents. To overcome these issues, we present a novel tetrazine scaffold-triazolyl-tetrazine-that can be readily synthesized from shelf-stable ethynyl-tetrazines and azides. Triazolyl-tetrazines exhibit improved physiological stability along with high reactivity. We showcase the effectiveness of this approach by creating cell-permeable probes for protein labeling and live cell imaging, as well as efficiently producing 18F-labeled molecular probes for positron emission tomography imaging. By utilizing the readily available pool of functionalized azides, we envisage that this modular approach will provide universal accessibility to tetrazine bioorthogonal tools, facilitating applications in biomedicine and materials science.

Fluorescent fatty acid conjugates for live cell imaging of peroxisomes

Peroxisomes are eukaryotic organelles that are essential for multiple metabolic pathways, including fatty acid oxidation, degradation of amino acids, and biosynthesis of ether lipids. Consequently, peroxisome dysfunction leads to pediatric-onset neurodegenerative conditions, including Peroxisome Biogenesis Disorders (PBD). Due to the dynamic, tissue-specific, and context-dependent nature of their biogenesis and function, live cell imaging of peroxisomes is essential for studying peroxisome regulation, as well as for the diagnosis of PBD-linked abnormalities. However, the peroxisomal imaging toolkit is lacking in many respects, with no reporters for substrate import, nor cell-permeable probes that could stain dysfunctional peroxisomes. Here we report that the BODIPY-C12 fluorescent fatty acid probe stains functional and dysfunctional peroxisomes in live mammalian cells. We then go on to improve BODIPY-C12, generating peroxisome-specific reagents, PeroxiSPY650 and PeroxiSPY555. These probes combine high peroxisome specificity, bright fluorescence in the red and far-red spectrum, and fast non-cytotoxic staining, making them ideal tools for live cell, whole organism, or tissue imaging of peroxisomes. Finally, we demonstrate that PeroxiSPY enables diagnosis of peroxisome abnormalities in the PBD CRISPR/Cas9 cell models and patient-derived cell lines.

Development of Inhibitors, Probes, and PROTAC Provides a Complete Toolbox to Study PARK7 in the Living Cell.

The integration of diverse chemical tools like small-molecule inhibitors, activity-based probes (ABPs), and proteolysis targeting chimeras (PROTACs) advances clinical drug discovery and facilitates the exploration of various biological facets of targeted proteins. Here, we report the development of such a chemical toolbox for the human Parkinson disease protein 7 (PARK7/DJ-1) implicated in Parkinson's disease and cancers. By combining structure-guided design, miniaturized library synthesis, and high-throughput screening, we identified two potent compounds, JYQ-164 and JYQ-173, inhibiting PARK7 in vitro and in cells by covalently and selectively targeting its critical residue, Cys106. Leveraging JYQ-173, we further developed a cell-permeable Bodipy probe, JYQ-196, for covalent labeling of PARK7 in living cells and a first-in-class PARK7 degrader JYQ-194 that selectively induces its proteasomal degradation in human cells. Our study provides a valuable toolbox to enhance the understanding of PARK7 biology in cellular contexts and opens new opportunities for therapeutic interventions.

Abstract 2044: A live cell PRMT5 NanoBRET Target Engagement Assay querying competitive and uncompetitive modes of inhibition

Abstract PRMT5 is an essential arginine methyltransferase that regulates a wide spectrum of cellular processes through the methylation of histone and non-histone substrates. In PRMT5 catalysis, SAM serves as the cofactor and methyl group donor, generating a methylated guanidinium moiety on the target substrate. In normal cells, the SAM pools are maintained through the methionine salvage pathway. In 15% of cancers, a key enzyme in this pathway (MTAP) is deleted, leading to an accumulation of the intermediate MTA, which inhibits PRMT5 activity. This genetic loss of function has therefore been pursued as a collateral vulnerability in MTAP deleted cancers. To exploit this synthetic lethality, a number of novel inhibitors have been developed that bind cooperatively at the PRMT5/MTA complex, offering a compelling pathway to precision medicine. Here we describe a novel, live cell NanoBRET Target Engagement assay that enables mechanistic studies on a variety of PRMT5 inhibitors. Using a cell-permeable NanoBRET probe directed to the substrate pocket of PRMT5, both substrate- and cofactor-competitive engagement can be quantified in cells. Moreover, this method can be used to quantify MTA-uncompetitive target engagement in cells, providing a platform to measure the potency of PRMT5-MTA-drug ternary complex formation. This method serves as a first-in-class method to quantify uncompetitive target engagement in live cells, which can be applied to other model systems. Citation Format: Ani Michaud, Elisabeth Rothweiler, Cesear Corona, Michael Beck, Jennifer Wilkinson, James Vasta, Kilian Huber, Matthew Robers. A live cell PRMT5 NanoBRET Target Engagement Assay querying competitive and uncompetitive modes of inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2044.

A cell-permeable Ub-Dha probe for profiling E1-E2-E3 enzymes in live cells.

Activity-based ubiquitin probes (Ub-ABPs) have recently been developed as effective tools for studying the capabilities of E1-E2-E3 enzymes, but most of them can only be used in cell lysates. Here, we report the first cell-penetrating Ub-Dha probes based on thiazolidine-protected cysteines, which enable successful delivery into cells confirmed by a fluorophore at the N-terminus of Ub and live-cell fluorescence microscopy. A total of 18 E1-E2-E3 enzymes in live cells were labelled and enriched in combination with label-free quantification (LFQ) mass spectrometry. This work provided a new cell-penetrating Ub tool for studying the activity and function of Ub-related enzymes.

A cell-permeable probe for the labelling of a bacterial glycosyltransferase and virulence factor.

Chemical probes for bacterial glycosyltransferases are of interest for applications such as tracking of expression levels, and strain profiling and identification. Existing probes for glycosyltransferases are typically based on sugar-nucleotides, whose charged nature limits their applicability in intact cells. We report the development of an uncharged covalent probe for the bacterial galactosyltransferase LgtC, and its application for the fluorescent labelling of this enzyme in recombinant form, cell lysates, and intact cells. The probe was designed by equipping a previously reported covalent LgtC inhibitor based on a pyrazol-3-one scaffold with a 7-hydroxycoumarin fluorophore. We show that this pyrazol-3-ones scaffold is surprisingly stable in aqueous media, which may have wider implications for the use of pyrazol-3-ones as chemical probes. We also show that the 7-hydroxycoumarin fluorophore leads to an unexpected improvement in activity, which could be exploited for the development of second generation analogues. These results will provide a basis for the development of LgtC-specific probes for the detection of LgtC-expressing bacterial strains.

Single Atom Stabilization of Phosphinate Ester-Containing Rhodamines Yields Cell Permeable Probes for Turn-On Photoacoustic Imaging.

Photoacoustic imaging (PAI) is an emerging imaging technique that uses pulsed laser excitation with near-infrared (NIR) light to elicit local temperature increases through non-radiative relaxation events, ultimately leading to the production of ultrasound waves. The classical xanthene dye scaffold has found numerous applications in fluorescence imaging, however, xanthenes are rarely utilized for PAI since they do not typically display NIR absorbance. Herein, we report the ability of Nebraska Red (NR) xanthene dyes to produce photoacoustic (PA) signal and provide a rational design approach to reduce the hydrolysis rate of ester containing dyes, affording cell permeable probes. To demonstrate the utility of this approach, we construct the first cell permeable rhodamine-based, turn-on PAI imaging probe for hypochlorous acid (HOCl) with maximal absorbance within the range of commercial PA instrumentation. This probe, termed SNR700 -HOCl, is capable of detecting exogenous HOCl in mice. This work provides a new set of rhodamine-based PAI agents as well as a rational design approach to stabilize esterified versions of NR dyes with desirable properties for PAI. In the long term, the reagents described herein could be utilized to enable non-invasive imaging of HOCl in disease-relevant model systems.

Cell-Permeable Stimuli-Responsive Ubiquitin Probe for Time-Resolved Monitoring of Substrate Ubiquitination in Live Cells.

Dynamic monitoring of intracellular ubiquitin (Ub) conjugates is instrumental to understanding the Ub regulatory machinery. Although many biochemical approaches have been developed to characterize protein ubiquitination, chemical tools capable of temporal resolution probing of ubiquitination events remain to be developed. Here, we report the development of the first cell-permeable and stimuli-responsive Ub probe and its application for the temporal resolution profiling of ubiquitinated substrates in live cells. The probe carrying the photolabile group N-(2-nitrobenzyl)-Gly (Nbg) on the amide bond between Ub Gly75 and Gly76 is readily prepared through chemical synthesis and can be delivered to live cells by conjugation via a disulfide bond with the cyclic cell-penetrating peptide cR10D (i.e., 4-((4-(dimethylamino)phenyl)-azo)-benzoic acid-modified cyclic deca-arginine). Both in vitro and in vivo experiments showed that Ub-modifying enzymes (E1, E2s, and E3s) could not install the Ub probe onto substrate proteins prior to removal of the nitrobenzyl group, which was easily accomplished via photoirradiation. The utility and practicality of this probe were exemplified by the time-resolved biochemical and proteomic investigation of ubiquitination events in live cells during a H2O2-mediated oxidative stress response. This work shows a conceptually new family of chemical Ub tools for the time-resolved studies on dynamic protein ubiquitination in different biological processes and highlights the utility of modern chemical protein synthesis in obtaining custom-designed tools for biological studies.

The dual effects of Congea chinensis Moldenke on inhibiting tumor cell proliferation and delaying aging by activating TERT transcriptional activity

Ethnopharmacological relevanceNatural medicinal plants, also named herbs, have attracted considerable research attention for their potential pharmacological activities, such as antitumor and longevity-promoting activities. Our previous review proposed that maintaining the homeostatic balance between aging and cancer may benefit organisms to enable tumor-free longevity. Congea chinensis Moldenke (CCM) is a plant species that grows on the border of Yunnan Province of China. Its medicinal value has been few reports until now. Thus, screening and extraction the ingredients from CCM that are both active tumor suppressors and TERT activators is a therapeutic strategy for improving tumor-free longevity. Aim of the studyTo extract and evaluate the cytotoxic antitumor and TERT transcription-promoting activities of the plant CCM. Materials and methodsThe ingredients extracted from CCM were tested for transcriptional activation of p53 using pGL4-p53-GFP cells and for TERT expression using a real-time PCR assay. In vitro antitumor activity was detected by sulforhodamine B (SRB) assay and Annexin V/PI staining assay. The cell-permeable probe H2DCFDA was used to detect intracellular reactive oxygen species (ROS). Western blot was performed to verify predicated proteins regulated by the ingredients. RNA-sequence analysis was applied to predicate the underlying mechanism of CCM. ResultsBoth CCM and MPRC2-8, two novel extracts of Congea chinensis Moldenke, activated the expression of p53 and TERT and were selectively cytotoxic toward tumor cells. In addition, the cytotoxic mechanism of MPRC2-8 was identified as ROS generation-induced apoptosis. Interestingly, MPRC2-8 showed opposite regulatory effects on the SIRT1-p53 axis in A549 and HT-29 cells, which have different p53 statuses. RNA-seq analysis showed that CCM and MPRC2-8 induced the p53, apoptosis and ROS signaling pathways, consistent with the results of cellular experiments in vitro. ConclusionOur study reveals that CCM and MPRC2-8 have two complementary activities, antitumor activity and TERT-activating activity, with potential antitumor and longevity-improving effects.

Development of a cell-permeable adenine-derived probe for capture of nucleotide-binding proteins in living cells

Adenine is an essential building block of genetic material and a range of coenzymes. Chemical probes containing an adenine moiety have been used in kinase profiling studies in cell lysates. Here we report that adenine-derived small-molecule probes with an activated ester reactive group can covalently modify a conserved lysine residue of protein kinases and capture a number of nucleotide-binding proteins within living cells.

A genetically encoded photoproximity labeling approach for mapping protein territories

Studying dynamic biological processes requires approaches compatible with the lifetimes of the biochemical transactions under investigation, which can be very short. We describe a genetically encoded system that allows protein neighborhoods to be mapped using visible light. Our approach involves fusing an engineered flavoprotein to a protein of interest. Brief excitation of the fusion protein leads to the labeling of nearby proteins with cell-permeable probes. Mechanistic studies reveal different labeling pathways are operational depending on the nature of the exogenous probe that is employed. When combined with quantitative proteomics, this photoproximity labeling system generates "snapshots" of protein territories with high temporal and spatial resolution. The intrinsic fluorescence of the fusion domain permits correlated imaging and proteomics analyses, a capability that is exploited in several contexts, including defining the protein clients of the major vault protein. The technology should be broadly useful in the biomedical area.

Abstract 4778: Novel bioluminescence approaches for measuring fatty acid β-oxidation and production of reactive oxygen and nitrogen species

Abstract Energy uptake and utilization in eukaryotic cells is a dynamic process regulated by a series of interacting metabolic networks. Interrogation of this complex network relies on rapid, sensitive approaches that do not require extensive sample handling and are easily adaptable to 96- and 384-well plates. Previously, using the Ultra-Glo luciferase reaction we developed a panel of bioluminescent assays that can be used to monitor numerous aspects of cellular metabolism and mitochondrial function including ATP production, glucose and amino acid metabolism, and the TCA cycle. Here, we extend the use of the luciferase reaction and report on the development of novel bioluminescence probes for studying two important metabolic cellular responses: fatty acid β-oxidation (FAO) and production of reactive oxygen and nitrogen species. For studying FAO, we developed a cell permeable probe with caged-luciferin attached to a fatty acid chain. The probe enters the cells and following FAO cycling, free luciferin is released and detected using Luciferin Detection Reagent. We validated the approach using known FAO activators and inhibitors and used it to monitor the changes in FAO during T-cell activation. For measuring reactive oxygen and nitrogen species, highly selective bioluminescence probes were developed. Upon reaction of the probes with their corresponding ROS target, they form the stable D-luciferin reporter molecule, causing luciferin to accumulate. Upon treatment with luciferase in the detection step, the generated light allows for quantification of the superoxide or nitric oxide formed. Both probes are suitable for in vitro and cell-based detection of ROS in an “add and read” format, providing for a simple workflow amenable to high throughput experimentation. Citation Format: Kim Haupt, Matt Larsen, Hui Wang, Natasha Karassina, Mike Valley, Wenhui Zhou, Jolanta Vidugiriene. Novel bioluminescence approaches for measuring fatty acid β-oxidation and production of reactive oxygen and nitrogen species. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4778.

In situ global proteomics profiling of EGCG targets using a cell-permeable and Click-able bioorthogonal probe

Despite possessing a wide spectrum of biological activities, molecular targets of EGCG remain elusive and as a result, its precise mode of action is still unknown. Herein, we have developed a novel cell-permeable and Click-able bioorthogonal probe for EGCG, YnEGCG for in situ detection and identification of its interacting proteins. The strategic structural modification on YnEGCG allowed it to retain innate biological activities of EGCG (IC50 59.52 ± 1.14 μM and 9.07 ± 0.01 μM for cell viability and radical scavenging activity, respectively). Chemoproteomics profiling identified 160 direct EGCG targets, with H:L ratio ≥ 1.10 from the list of 207 proteins, including multiple new proteins that were previously unknown. The targets were broadly distributed in various subcellular compartments suggesting a polypharmacological mode of action of EGCG. GO analysis revealed that the primary targets belonged to the enzymes that regulate key metabolic processes including glycolysis and energy homeostasis, also the cytoplasm (36 %) and mitochondria (15.6 %) contain the majority of EGCG targets. Further, we validated that EGCG interactome was closely associated with apoptosis indicating its role in inducing toxicity in cancer cells. For the first time, this in situ chemoproteomics approach could identify a direct and specific EGCG interactome under physiological conditions in an unbiased manner.

De Novo Designed Self-Assembling Rhodamine Probe for Real-Time, Long-Term and Quantitative Live-Cell Nanoscopy.

Super-resolution imaging provides a powerful approach to image dynamic biomolecule events at nanoscale resolution. An ingenious method involving tuning intramolecular spirocyclization in rhodamine offers an appealing strategy to design cell-permeable fluorogenic probes for super-resolution imaging. Nevertheless, precise control of rhodamine spirocyclization presents a significant challenge. Through detailed study of the structure-activity relationship, we identified that multiple key factors control rhodamime spirocyclization. The findings provide opportunities to create fluorogenic probes with tailored properties. On the basis of our findings, we constructed self-assembling rhodamine probes for no-wash live-cell confocal and super-resolution imaging. The designed self-assembling probe Rho-2CF3 specifically labeled its target proteins and displayed high ring-opening ability, fast labeling kinetics (<1 min), and large turn-on fold (>80 folds), which is very difficult to be realized by the existing methods. Using the probe, we achieved high-contrast super-resolution imaging of nuclei and mitochondria with a spatial resolution of up to 42 nm. The probe also showed excellent photostability and proved ideal for real-time and long-term tracking of mitochondrial fission and fusion events with high spatiotemporal resolution. Furthermore, Rho-2CF3 could resolve the ultrastructure of mitochondrial cristae and quantify their morphological changes under drug treatment at nanoscale. Our strategy thus demonstrates its usefulness in designing self-assembling probes for super-resolution imaging.

Solanum dulcamara L. Berries: A Convenient Model System to Study Redox Processes in Relation to Fruit Ripening.

The present study provides, for the first time, a physicochemical and biochemical characterization of the redox processes associated with the ripening of Solanum dulcamara L. (bittersweet) berries. Electron Paramagnetic Resonance Spectroscopy (EPRS) and Imaging (EPRI) measurements of reactive oxygen species (ROS) were performed in parallel with the tissue-specific metabolic profiling of major antioxidants and assessment of antioxidant enzymes activity. Fruit transition from the mature green (MG) to ripe red (RR) stage involved changes in the qualitative and quantitative content of antioxidants and the associated cellular oxidation and peroxidation processes. The skin of bittersweet berries, which was the major source of antioxidants, exhibited the highest antioxidant potential against DPPH radicals and nitroxyl spin probe 3CP. The efficient enzymatic antioxidant system played a critical protective role against the deleterious effects of progressive oxidative stress during ripening. Here, we present the EPRI methodology to assess the redox status of fruits and to discriminate between the redox states of different tissues. Interestingly, the intracellular reoxidation of cell-permeable nitroxide probe 3CP was observed for the first time in fruits or any other plant tissue, and its intensity is herein proposed as a reliable indicator of oxidative stress during ripening. The described noninvasive EPRI technique has the potential to have broader application in the study of redox processes associated with the development, senescence, and postharvest storage of fruits, as well as other circumstances in which oxidative stress is implicated.

Development of an activity-based chemiluminogenic probe for γ-glutamylcyclotransferase.

While γ-glutamylcyclotransferase (GGCT) has been implicated in cancer-cell proliferation, the role of GGCT enzymatic activity in the regulation of cancer-cell growth remains unclear. Toward further understanding of GGCT in vivo, here we report a novel cell-permeable chemiluminogenic probe "MAM-LISA-103" that detects intracellular GGCT activity and apply it to in vivo imaging. We first developed a chemiluminogenic probe LISA-103, which simply and sensitively detects the enzymatic activity of recombinant GGCT through chemiluminescence. We then designed the cell-permeable GGCT probe MAM-LISA-103 and applied it to several biological experiments. MAM-LISA-103 successfully detected the intracellular GGCT activity in GGCT-overexpressing NIH-3T3 cells. Moreover, MAM-LISA-103 demonstrated tumor-imaging ability when administered to a xenograft model using immunocompromised mice inoculated with MCF7 cells.

Antiplasmodial Properties of Aqueous and Ethanolic Extracts of Ten Herbal Traditional Recipes Used in Thailand against Plasmodium falciparum.

This study evaluated the in vitro and in vivo antiplasmodial efficacy and toxicity of aqueous and ethanolic extracts from traditional recipes used in Thailand. The aqueous and ethanolic extracts of ten traditional recipes were tested for in vitro antiplasmodial activity (parasite lactate dehydrogenase assay), cytotoxicity (MTT assay), and hemolysis). Oxidant levels were measured using cell-permeable probe 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate fluorescent dye-based assays. The best candidate was chosen for testing in mouse models using 4-day suppressive and acute toxicity assays. An in vitro study showed that ethanolic extracts and three aqueous extracts exhibited antiplasmodial activity, with an IC50 in the range of 2.8-15.5 µg/mL. All extracts showed high CC50 values, except for ethanolic extracts from Benjakul, Benjalotiga, and Trikatuk in HepG2 and Benjalotiga and aqueous extract from Chan-tang-ha in a Vero cell. Based on the results of the in vitro antiplasmodial activity, an aqueous extract of Triphala was chosen for testing in mouse models. The aqueous extract of Triphala exhibited good antiplasmodial activity, was safe at an oral dose of 2 g/kg, and is a potential candidate as a new source for the development of antimalarial drugs.

Histochemical targeting: Combining plasma immersion ion implantation and histochemical probes to target magnetic particles to intracellular cytological features

Paramagnetic microparticles are effective tools for cell purification, diagnostics, imaging and drug delivery and have been used for over 30 years to separate cell types from complex mixtures. The targeting of magnetic particles to cells and tissues is dependent upon the use of antibodies and adhesion molecules as targeting modalities. Here, we present a novel paradigm for the targeting of microparticles to cells according to intracellular cytological characteristics. Cell-permeable histochemical dyes, when covalently conjugated to magnetic microparticles, following plasma immersion ion implantation (PIII) imbue the particles with cell targeting properties in a process we term histochemical targeting. PIII treatment and conjugation to the lipophilic dyes Nile Red or Nile Blue led to changes in microparticle size, surface charge and lipophilicity. PIII-treated microparticles covalently conjugated with Nile Red, crossed the live cell membrane and homed to intracellular neutral lipid droplets. Nile Blue-conjugated particles crossed live cell membranes and remained in the cytosol where free fatty acids localize. Magnetic cell sorting could be performed on cultured adipocytes, as well as rat liver and adrenal cell suspensions using particles conjugated to Nile Red or Nile Blue. Taken together, our findings demonstrate that histochemical probes can be covalently conjugated to magnetic particles following PIII treatment resulting in intracellular targeting. With a vast armamentarium of cell-permeable probes available, histochemical targeting could be applied to many fields requiring magnetic particle delivery to specific sites or isolation of particular cell types.

Puerarin prevents calcium oxalate crystal-induced renal epithelial cell autophagy by activating the SIRT1-mediated signaling pathway.

Calcium oxalate (CaOx) crystals can activate autophagy, causing damage to renal tubular epithelial cells (TECs). Puerarin has been shown to have protective and therapeutic effects against a variety of diseases by inhibiting autophagy activation. However, the protective effect of puerarin against CaOx crystals and the underlying molecular mechanisms are unclear. Cell Counting Kit-8 (CCK-8) assays were used to evaluate the effects of puerarin on cell viability. Intracellular reactive oxygen species (ROS) levels were measured by the cell-permeable fluorogenic probe 2,7-dichlorofluorescein diacetate (DCFH-DA). Immunofluorescence, immunohistochemistry, and western blotting were used to examine the expression of SIRT1, Beclin1, p62, and LC3, and explore the underlying molecular mechanisms in vivo and in vitro. Puerarin treatment significantly attenuated CaOx crystal-induced autophagy of TECs and CaOx cytotoxicity to TECs by altering SIRT1 expression in vitro and in vivo, whereas the SIRT1-specific inhibitor EX527 exerted contrasting effects. In addition, we found that the protective effect of puerarin was related to the SIRT1/AKT/p38 signaling pathway. The findings suggest that puerarin regulates CaOx crystal-induced autophagy by activating the SIRT1-mediated signaling pathway, and they suggest a series of potential therapeutic targets and strategies for treating nephrolithiasis.