U2OS Cells Research Articles

Cell Painting and Chemical Structure Read-Across Can Complement Each Other for Rat Acute Oral Toxicity Prediction in Chemical Early Derisking.

Early derisking decisions in the development of new chemical compounds enable the identification of novel chemical candidates with improved safety profiles. In vivo studies are traditionally conducted in the early assessment of acute oral toxicity of crop protection products to avoid compounds, which are considered "very acutely toxic", with an in vivo lethal dose of 50% (LD50) ≤ 60 mg/kg body weight. Those studies are lengthy and costly and raise ethical concerns, catalyzing the use of nonanimal alternatives. The objective of our analysis was to assess the predictive efficacy of read-across approaches for acute oral toxicity in rats, comparing the use of chemical structure information, in vitro biological data derived from the Cell Painting profiling assay on U2OS cells, or the combination of both. Our findings indicate that the classification of compounds as very acute oral toxic (LD50 ≤ 60 mg/kg) or not is possible using a read-across approach, with chemical structure information, morphological profiles, or a combination of both. When classifying compounds structurally similar to those in the training set, the chemical structure was more predictive (balanced accuracy of 0.82). Conversely, when the compounds to be classified were structurally different from those in the training set, the morphological profiles were more predictive (balanced accuracy of 0.72). Combining the two models allowed for the classification of compounds structurally similar to those in the training set to slightly improve the predictions (balanced accuracy of 0.85).

Single-shot quantitative differential phase contrast microscope using a single calcite beam displacer

This paper presents the development of a single-shot, partially spatially coherent quantitative differential phase contrast microscopy (Q-DPCM) system. The optical scheme comprises a polarizer, lenses, calcite beam displacer, and analyzer, which can be seamlessly integrated to an existing bright-field microscopy system, transforming it into a Q-DPCM system. It utilizes a partially spatially coherent light source, enabling single-shot quantitative differential phase recovery of the specimens with high spatial phase sensitivity. It generates highly sensitive quantitative differential phase images of the specimens along one direction, like a gradient light interference microscopy (GLIM) system, using only a single interferogram. First, we validated the differential phase measurement capability of the system through experiments on polystyrene spheres (diameter 5.2 µm) and HeLa cells. Next, the system is utilized to generate quantitative phase maps of human red blood cells using two orthogonal differential interferograms recorded at two orientations of the calcite beam displacer. Further, the Q-DPCM system is implemented for 1-h time-lapse live cell monitoring, revealing the dynamics of intracellular granules such as nucleolus and lipids in U2OS cells.

Tanshinone II A Facilitates Chemosensitivity of Osteosarcoma Cells to Cisplatin via Activation of p38 MAPK Pathway.

To examine the mechanism of action of tanshinone II A (Tan II A) in promoting chemosensitization of osteosarcoma cells to cisplatin (DDP). The effects of different concentrations of Tan II A (0-80 µ mol/L) and DDP (0-2 µ mol/L) on the proliferation of osteosarcoma cell lines (U2R, U2OS, 143B, and HOS) at different times were examined using the cell counting kit-8 and colony formation assays. Migration and invasion of U2R and U2OS cells were detected after 24 h treatment with 30 µ mol/L Tan II A, 0.5 µ mol/L DDP alone, and a combination of 10 µ mol/L Tan II A and 0.25 µ mol/L DDP using the transwell assay. After 48 h of treatment of U2R and U2OS cells with predetermined concentrations of each group of drugs, the cell cycle was analyzed using a cell cycle detection kit and flow cytometry. After 48 h treatment, apoptosis of U2R and U2OS cells was detected using annexin V-FITC apoptosis detection kit and flow cytometry. U2R cells were inoculated into the unilateral axilla of nude mice and then the mice were randomly divided into 4 groups of 6 nude mice each. The 4 groups were treated with equal volume of Tan II A (15 mg/kg), DDP (3 mg/kg), Tan II A (7.5 mg/kg) + DDP (1.5 mg/kg), and normal saline, respectively. The body weight of the nude mice was weighed, and the tumor volume and weight were measured. Cell-related gene and signaling pathway expression were detected by RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analysis. p38 MAPK signaling pathway proteins and apoptotic protein expressions were detected by Western blot. In vitro studies have shown that Tan II A, DDP and the combination of Tan II A and DDP inhibit the proliferation, migration and invasion of osteosarcoma cells. The inhibitory effect was more pronounced in the Tan II A and DDP combined treatment group (P<0.05 or P<0.01). Osteosarcoma cells underwent significantly cell-cycle arrest and cell apoptosis by Tan II A-DDP combination treatment (P<0.05 or P<0.01). In vivo studies demonstrated that the Tan II A-DD combination treatment group significantly inhibited tumor growth compared to the Tan II A and DDP single drug group (P<0.01). Additionally, we found that the combination of Tan II A and DDP treatment enhanced the p38 MAPK signaling pathway. Western blot assays showed higher p-p38, cleaved caspase-3, and Bax and lower caspase-3, and Bcl-2 expressions with the combination of Tan II A and DDP treatment compared to the single drug treatment (P<0.01). Tan II A synergizes with DDP by activating the p38/MAPK pathway to upregulate cleaved caspase-3 and Bax pro-apoptotic gene expressions, and downregulate caspase-3 and Bcl-2 inhibitory apoptotic gene expressions, thereby enhancing the chemosensitivity of osteosarcoma cells to DDP.

Hundreds of myosin 10s are pushed to the tips of filopodia and could cause traffic jams on actin

Myosin 10 (Myo10) is a motor protein known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the absolute number of Myo10 molecules during the filopodial lifecycle. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. We combined SDS-PAGE densitometry with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it enriches at opposite cellular ends. Hundreds of Myo10s are in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Live-cell movies reveal a dense cluster of over a hundred Myo10 molecules that initiates filopodial elongation. Hundreds of Myo10 molecules continue to accumulate during filopodial growth, but accumulation ceases when retraction begins. Rates of filopodial elongation, second-phase elongation, and retraction are inversely related to Myo10 quantities. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane compartments. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation.

Hundreds of myosin 10s are pushed to the tips of filopodia and could cause traffic jams on actin.

Myosin 10 (Myo10) is a motor protein known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the absolute number of Myo10 molecules during the filopodial lifecycle. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. We combined SDS-PAGE densitometry with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it enriches at opposite cellular ends. Hundreds of Myo10s are in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Live-cell movies reveal a dense cluster of over a hundred Myo10 molecules that initiates filopodial elongation. Hundreds of Myo10 molecules continue to accumulate during filopodial growth, but accumulation ceases when retraction begins. Rates of filopodial elongation, second-phase elongation, and retraction are inversely related to Myo10 quantities. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane compartments. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation.

Dopamine internalization via Uptake2 and stimulation of intracellular D5-receptor-dependent calcium mobilization and CDP-diacylglycerol signaling

Dopamine stimulates CDP-diacylglycerol biosynthesis through D1-like receptors, particularly the D5 subtype most of which is intracellularly localized. CDP-diacylglycerol regulates phosphatidylinositol-4,5-bisphosphate-dependent signaling cascades by serving as obligatory substrate for phosphatidylinositol biosynthesis. Here, we used acute and organotypic brain tissues and cultured cells to explore the mechanism by which extracellular dopamine acts to modulate intracellular CDP-diacylglycerol. Dopamine stimulated CDP-diacylglycerol in organotypic and neural cells lacking the presynaptic dopamine transporter, and this action was selectively mimicked by D1-like receptor agonists SKF38393 and SKF83959. Dopaminergic CDP-diacylglycerol stimulation was blocked by decynium-22 which blocks Uptake2-like transporters and by anti-microtubule disrupters of cytoskeletal transport, suggesting transmembrane uptake and guided transport of the ligands to intracellular sites of CDP-diacylglycerol regulation. Fluorescent or radiolabeled dopamine was saturably transported into primary neurons or B35 neuroblastoma cells expressing the plasmamembrane monoamine transporter, PMAT. Microinjection of 10-nM final concentration of dopamine into human D5-receptor-transfected U2-OS cells rapidly and transiently increased cytosolic calcium concentrations by 316%, whereas non-D5-receptor-expressing U2-OS cells showed no response. Given that U2-OS cells natively express PMAT, bath application of 10 μM dopamine slowly increased cytosolic calcium in D5-expressing cells. These observations indicate that dopamine is actively transported by a PMAT-implicated Uptake2-like mechanism into postsynaptic-type dopaminoceptive cells where the monoamine stimulates its intracellular D5-type receptors to mobilize cytosolic calcium and promote CDP-diacylglycerol biosynthesis. This is probably the first demonstration of functional intracellular dopamine receptor coupling in neural tissue, thus challenging the conventional paradigm that postsynaptic dopamine uptake serves merely as a mechanism for deactivating spent or excessive synaptic transmitter.

Crosstalk between 1,25(OH)2-Vitamin D3 and the growth factors EGF and PDGF-BB: Impact on CYP24A1 expression and cell proliferation

This study explored the signaling interplay between the vitamin D receptor (VDR) and receptor tyrosine kinases (RTKs). Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-BB promotes cell proliferation in normal and cancer cells. At the same time, the active form of vitamin D (1,25(OH)2-vitamin D3) inhibits proliferation in some cells. Although EGF receptors (EGFR) and PDGF receptors (PDGFR) activate similar downstream pathways, we found that they interact with VDR signaling in distinct ways. We confirmed that 1,25(OH)2-vitamin D3 induces CYP24A1 gene expression in U2OS, T98G, and U251 cells. We found this to be potentiated when combined with EGF. In contrast, PDGF-BB did not impact 1,25(OH)2-vitamin D3-induced CYP24A1 expression in U2OS cells. The increase in CYP24A1 expression due to the combined action of EGF and 1,25(OH)2-vitamin D3 was dependent on AKT and ERK1/2 activation. Another VDR-responsive gene, CYP27B1, was unaffected by the addition of EGF, suggesting that EGF may have gene-specific effects on VDR signaling. While PDGF-BB did not influence CYP24A1 expression, 1,25(OH)2-vitamin D3 significantly influenced PDGF-BB-induced receptor phosphorylation and cell proliferation. In summary, we found that EGF, but not PDGF-BB, influenced the expression of the VDR-dependent gene CYP24A1, while 1,25(OH)2-vitamin D3 had an inhibitory effect on PDGFR signaling and proliferation. These findings highlight unique crosstalk between 1,25(OH)2-vitamin D3 signaling and EGF or PDGF-BB.

Novel platinum nanoclusters (Pt NCs) induce mitochondrial apoptosis and damaging autophagy for the treatment of osteosarcoma—from the perspective of P53 mutation status in different cell lines

This study aimed to investigate the anticancer efficacy and underlying mechanism of novel platinum nanoclusters (Pt NCs) in osteosarcoma cell lines exhibiting distinct P53 expression profiles, namely MG-63 (P53−) and U2-OS (P53+). The findings revealed that Pt NCs exerted an inhibitory effect on proliferation, migration, and colony formation while promoting apoptosis in both MG-63 (P53−) and U2-OS (P53+) cells. The inhibitory effect on the malignant characteristics of MG-63 (P53−) cells was more obvious, indicating that the potential anticancer effect of Pt NCs was not dependent on P53. Animal experiments have substantiated the in vivo anticancer properties of Pt NCs, while also revealing their lower toxicity on cells and tissues. Pt NCs possess the ability to impede cell proliferation by inducing DNA damage and arresting the cell cycle in the G1 phase and possess the ability to promote BAX/Bcl-2/Caspase-3/mitochondrial apoptosis. Pt NCs may promote mitochondrial apoptosis by promoting damaging autophagy, thereby promoting cellular demise. This study has confirmed the P53-independent anticancer impact of Pt NCs on osteosarcoma in vitro and in vivo. Pt NCs may play a therapeutic role in more sensitive MG-63 (P53−) cells by promoting DNA damage to arrest the cell cycle, stimulating BAX/Bcl-2/Caspase-3/mitochondrial apoptosis, and initiating damaging autophagy.

HIV-1 Vpr-induced DNA damage activates NF-κB through ATM-NEMO independent of cell cycle arrest.

Lentiviruses encode a number of multi-functional accessory proteins, however, the primary role of the accessory protein Vpr remains unclear. As Vpr engages the host DNA damage response (DDR) at multiple steps, modulation of the DDR is considered central to the function(s) of Vpr. Vpr activates ataxia telangiectasia and Rad3 (ATR)-mediated DDR signaling, resulting in cell cycle arrest. However, the cellular consequences of Vpr-induced DNA damage, and the connection of Vpr-induced DNA damage to other Vpr functions, are unknown. Here, we determined that HIV-1 Vpr-induced DNA damage activates the ATM-NF-κB essential modulator (NEMO) pathway and alters cellular transcription via NF-κB/RelA. Through RNA-sequencing (RNA-seq) of cells expressing Vpr or mutants that separate the ability of Vpr to induce DNA damage from other DDR phenotypes, we identified that Vpr alters the transcriptome independent of cell cycle arrest. In tissue-cultured U2OS cells and primary human monocyte-derived macrophages (MDMs), we showed Vpr activates both ataxia telangiectasia mutated (ATM) and NF-κB/RelA signaling cascades. While inhibition of NEMO did not affect Vpr-induced DNA damage, it prevented NF-κB activation by Vpr, highlighting the importance of NEMO in Vpr-mediated transcriptional reprogramming. Virion-delivered Vpr was sufficient to induce DNA damage and activate ATM-NEMO dependent NF-κB transcription, suggesting that engagement of the DDR and transcriptional changes can occur early during viral replication. Together, our data uncover cellular consequences of Vpr-induced DNA damage and provide a mechanism for how Vpr activates NF-κB through DNA damage and ATM-NEMO signaling, which occur independent of cell cycle arrest. We propose this is essential to overcoming restrictive environments, such as in macrophages, to enhance viral replication.IMPORTANCEThe HIV accessory protein Vpr is multi-functional and required for viral replication in vivo, yet how Vpr enhances viral replication is unknown. Emerging literature suggests that a conserved function of Vpr is the engagement of the host DNA damage response (DDR). For example, Vpr activates DDR signaling, causes DDR-dependent cell cycle arrest, promotes degradation of various DDR proteins, and alters cellular consequences of DDR activation. However, a central understanding of how these phenotypes connect and how they affect HIV-infected cells remains unknown. Here, we found that Vpr-induced DNA damage alters the host transcriptome by activating an essential transcription pathway, NF-κB. This occurs early during the infection of primary human immune cells, suggesting NF-κB activation and transcriptome remodeling are important for establishing productive HIV-1 infection. Together, our study provides novel insights into how Vpr alters the host environment through the DDR, and what roles Vpr and the DDR play to enhance HIV replication.

ESOMA-DM1, a Maytansinoid-Based Theranostic Small-Molecule Drug Conjugate for Neuroendocrine Tumors.

Background: The main challenges of conventional chemotherapy lie in its lack of selectivity and specificity, leading to significant side effects. Using a small-molecule drug conjugate (SMDC) ensures specific delivery of a cytotoxic drug to the tumor site by coupling it to a targeting vector. This promising strategy can be applied to neuroendocrine tumors (NETs) by choosing a targeting vector that binds specifically to somatostatin receptor subtype 2 (SSTR2). Additionally, incorporation of a bifunctional chelate into the molecule enables complexation of both diagnostic and therapeutic radionuclides. Thus, it facilitates monitoring of the distribution of the SMDC in the body and allows for the implementation of combination therapy. In our study, we designed eSOMA-DM1, a SMDC combining the SSTR2-targeted octreotate peptide and the cytotoxic agent DM1 via a chelate-bridged linker (N3-Py-DOTAGA). This approach warrants conjugation of the targeting vector and the drug at opposite sites to avoid undesired steric hindrance effects. Methods: Synthesis of the DM1 moiety (4) involved a three-step synthetic route, followed by the conjugation to the cyclic peptide, N3-Py-DOTAGA-d-Phe-cyclo[Cys-Tyr-d-Trp-Lys-Thr-Cys]-Thr-OH, through a copper-free click reaction, resulting in eSOMA-DM1. Subsequent labeling with [111In]InCl3 gave a high radiochemical yield and purity. In vitro assessments of eSOMA-DM1 binding, uptake, and internalization were conducted in SSTR2-transfected U2OS cells. Ex vivo biodistribution and fluorescence imaging were performed in H69-tumor bearing mice. Results: eSOMA-DM1 exhibited an IC50 value for SSTR2 similar to the gold standard DOTA-TATE. The uptake of [111In]In-eSOMA-DM1 in U2OS.SSTR2 cells was 1.2-fold lower than that of [111In]In-DOTA-TATE. Tumor uptake in H69-xenografted mice was higher for [111In]In-eSOMA-DM1 at all-time points compared to [111In]In-DOTA-TATE. Prolonged blood circulation led to increased accumulation of [111In]In-eSOMA-DM1 in highly vascularized tissues, such as the lungs, skin, and heart. Excretion through the kidneys, liver, and spleen was also observed. Conclusion: eSOMA-DM1 is a SMDC developed for NET showing promising characteristics in vitro. However, the in vivo results obtained with [111In]In-eSOMA-DM1 suggest the need for adjustments to optimize its distribution.

Maneuvering the mineralization of self-assembled peptide nanofibers for designing mechanically-stiffened self-healable composites toward bone-mimetic ECM.

Extracellular matrix (ECM) elasticity remains a crucial parameter to determine cell-material interactions (viz. adhesion, growth, and differentiation), cellular communication, and migration that are essential to tissue repair and regeneration. Supramolecular peptide hydrogels with their 3-dimensional porous network and tuneable mechanical properties have emerged as an excellent class of ECM-mimetic biomaterials with relevant dynamic attributes and bioactivity. Here, we demonstrate the design of minimalist amyloid-inspired peptide amphiphiles, CnPA (n = 6, 8, 10, 12) with tuneable peptide nanostructures that are efficiently biomineralized and cross-linked using bioactive silicates. Such hydrogel composites, CnBG exhibit excellent mechanical attributes and possess excellent self-healing abilities and collagen-like strain-stiffening ability as desired for bone ECM mimetic scaffold. The composites exhibited the formation of a hydroxyapatite mineral phase upon incubation in a simulated body fluid that rendered mechanical stiffness akin to the hydroxyapatite-bridged collagen fibers to match the bone tissue elasticity eventually. In a nutshell, peptide nanostructure-guided temporal effects and mechanical attributes demonstrate C8BG to be an optimal composite. Finally, such constructs feature the potential for adhesion, proliferation of U2OS cells, high alkaline phosphatase activity, and osteoconductivity.

The FAM114A proteins are adaptors for the recycling of Golgi enzymes.

Golgi-resident enzymes remain in place while their substrates flow through from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the Golgi to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and one COPI adaptor protein, GOLPH3, acts to recruit enzymes into vesicles in part of the stack. Here, we used proximity biotinylation to identify further components of intra-Golgi vesicles and found FAM114A2, a cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1, showed that they bind to Golgi-resident membrane proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not cause substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Drosophila mutants lacking FAM114A have defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins bind Golgi enzymes and are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack.

ANK1 inhibits malignant progression of osteosarcoma by promoting ferroptosis

PurposeOsteosarcoma (OS) is a primary bone tumor with high malignancy and poor prognosis. Ferroptosis plays a crucial role in OS. This study aimed to evaluate the effects of Ankyrin 1 (ANK1) on OS and to investigate its specific mechanisms.MethodsMicroarray datasets related to “osteosarcoma” were selected for this study. Relevant hub genes in OS were identified through bioinformatics analysis. Transfected U-2OS and MG-63 cells were used for in vitro experiments. The effects of ANK1 overexpression on cell viability, migration, and invasion were determined through CCK-8, wound healing, and transwell assays. An OS mouse model was established for the in vivo experiments. Hematoxylin-eosin staining and immunohistochemistry were conducted to observe the histological effects of ANK1 overexpression on mouse tumors. TUNEL staining was performed to evaluate apoptosis in mouse.ResultsThere were 159 common differentially expressed genes in the GSE16088 and GSE19276 datasets. The hub genes ANK1, AHSP, GYPB, GYPA, KEL, and ALAS2 were identified. Pan-cancer analysis verified that ANK1 was closely associated with cancer prognosis and immune infiltration. Furthermore, ANK1 overexpression inhibited the proliferation, migration, and invasion of OS cells and promoted ferroptosis, while ferroptosis inhibitor (fer-1) weakened these effects. Moreover, ANK1 overexpression suppressed tumor growth, promoted apoptosis, reduced the number of Ki67 positive cells, and elevated the number of caspase-3 positive cells in vivo.ConclusionsANK1 is a prognosis biomarker of OS that can alleviate the progression of OS by promoting ferroptosis.

Human genomic DNA is widely interspersed with i-motif structures.

DNA i-motif structures are formed in the nuclei of human cells and are believed to provide critical genomic regulation. While the existence, abundance, and distribution of i-motif structures in human cells has been demonstrated and studied by immunofluorescent staining, and more recently NMR and CUT&Tag, the abundance and distribution of such structures in human genomic DNA have remained unclear. Here we utilise high-affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in the purified genomic DNA of human MCF7, U2OS and HEK293T cells. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach aimed to identify DNA sequences capable of i-motif formation on a genome-wide scale, revealing that such sequences are widely distributed throughout the human genome and are common in genes upregulated in G0/G1 cell cycle phases. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA and a foundational resource for future studies of their genomic, structural, and molecular roles.

Multi-omics correlates of insulin resistance and circadian parameters mapped directly from human serum.

While it is generally known that metabolic disorders and circadian dysfunction are intertwined, how the two systems affect each other is not well understood, nor are the genetic factors that might exacerbate this pathological interaction. Blood chemistry is profoundly changed in metabolic disorders, and we have previously shown that serum factors change cellular clock properties. To investigate if circulating factors altered in metabolic disorders have circadian modifying effects, and whether these effects are of genetic origin, we measured circadian rhythms in U2OS cell in the presence of serum collected from diabetic, obese or control subjects. We observed that circadian period lengthening in U2OS cells was associated with serum chemistry that is characteristic of insulin resistance. Characterizing the genetic variants that altered circadian period length by genome-wide association analysis, we found that one of the top variants mapped to the E3 ubiquitin ligase MARCH1 involved in insulin sensitivity. Confirming our data, the serum circadian modifying variants were also enriched in type 2 diabetes and chronotype variants identified in the UK Biobank cohort. Finally, to identify serum factors that might be involved in period lengthening, we performed detailed metabolomics and found that the circadian modifying variants are particularly associated with branched chain amino acids, whose levels are known to correlate with diabetes and insulin resistance. Overall, our multi-omics data showed comprehensively that systemic factors serve as a path through which metabolic disorders influence circadian system, and these can be examined in human populations directly by simple cellular assays in common cultured cells.

PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients.

PAK4 and PD-L1 have been suggested as novel therapeutic targets in human cancers. Moreover, PAK4 has been suggested to be a molecule closely related to the immune evasion of cancers. Therefore, this study evaluated the roles of PAK4 and PD-L1 in the progression of osteosarcomas in 32 osteosarcomas and osteosarcoma cells. In human osteosarcomas, immunohistochemical positivity for the expression of PAK4 (overall survival, p = 0.028) and PD-L1 (relapse-free survival, p = 0.002) were independent indicators for the survival of patients in a multivariate analysis. In osteosarcoma cells, the overexpression of PAK4 increased proliferation and invasiveness, while the knockdown of PAK4 suppressed proliferation and invasiveness. The expression of PAK4 was associated with the expression of the molecules related to cell cycle regulation, invasion, and apoptosis. PAK4 was involved in resistance to apoptosis under a treatment regime with doxorubicin for osteosarcoma. In U2OS cells, PAK4 was involved in the stabilization of PD-L1 from ubiquitin-mediated proteasomal degradation and the in vivo infiltration of immune cells such as regulatory T cells and PD1-, CD4-, and CD8-positive cells in mice tumors. In conclusion, this study suggests that PAK4 is involved in the progression of osteosarcoma by promoting proliferation, invasion, and resistance to doxorubicin and stabilized PD-L1 from proteasomal degradation.

Diazirine-AIOC-NAADP, a Clickable-Photoactive NAADP Analog for Sea Urchin NAADP Binding Proteins.

Calcium ions (Ca2+) play a vital role as intracellular messengers, regulating essential cellular processes. Nicotinic acid adenine dinucleotide phosphate (NAADP) serves as a potent second messenger, responsible for releasing Ca2+ in both mammals and echinoderms. Despite identification of two human NAADP receptor proteins, their counterparts in sea urchins remain elusive. Sea urchin NAADP binding proteins are important due to their unique identities and NAADP binding properties which may illuminate new signaling modalities in other species. Consequently, the development of new photoactive and clickable NAADP analogs with specificity for binding targets in sea urchin egg homogenates is a priority. We designed and synthesized diazirine-AIOC-NAADP, a photoactive and "clickable" NAADP analog, to specifically label and identify sea urchin NAADP receptors. This analog, synthesized using a chemo-enzymatic approach, induced Ca2+ release from sea urchin egg homogenates at low-micromolar concentrations. The ability of diazirine-AIOC-NAADP to mobilize Ca2+ in cultured human cells was investigated by microinjection of the probe into U2OS cells. Microinjected NAADP elicited a robust Ca2+ release, but even 6000-fold higher concentrations of diazirine-AIOC-NAADP were unable to release Ca2+. Our results indicate that our new probe is specifically recognized at low concentration by sea urchin egg NAADP receptors but not by the NAADP receptors in a human cultured cell line.

Hundreds of myosin 10s are pushed to the tips of filopodia and could cause traffic jams on actin.

Myosin 10 (Myo10) is a motor protein known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the absolute number of Myo10 molecules during the filopodial lifecycle. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. We combined SDS-PAGE densitometry with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it enriches at opposite cellular ends. Hundreds of Myo10s are in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Live-cell movies reveal a dense cluster of over a hundred Myo10 molecules that initiates filopodial elongation. Hundreds of Myo10 molecules continue to accumulate during filopodial growth, but accumulation ceases when retraction begins. Rates of filopodial elongation, second-phase elongation, and retraction are inversely related to Myo10 quantities. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane compartments. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation.

Investigation of Electrical Impedance of Human Breast Cancer Cells and Osteosarcoma Cells

Nanostructures exhibit a high aspect ratio and volume-specific surface area, endowing them with unique physical and chemical properties distinct from conventional bulk materials[1]. This study aims to develop cell-based biosensors (CBBs) for the non-invasive monitoring of MCF-7 (ATCC-HTB-22) and U2OS (ATCC-HTB-96) cell conditions. Vertically aligned nanostructures, coated with indium tin oxide (ITO), were employed as sensing substrates for cell growth. A polystyrene cylinder, attached to the (3-aminopropyl) trimethoxysilane (APTMS)-modified ITO sensing substrate using PDMS, was utilized to create a cell culture chamber. Impedance measurements were conducted with an E4980A precision LCR meter (Agilent Technologies) at 1 V, covering a frequency range of 20 Hz to 2 MHz, with 201 sample points. We verified the establishment of a functional biointerface by seeding MCF-7 and U2OS cells and monitoring impedance changes, indicative of cell attachment and growth. In conclusion, we successfully adopted the APTMS-modified ITO-coated sensing substrate to measure the impedance of MCF-7 and U2OS cells. Our goal is for this CBB platform to significantly contribute to the advancement of cancer drug development. References A. Gao et al., "Silicon-Nanowire-Based CMOS-Compatible Field-Effect Transistor Nanosensors for Ultrasensitive Electrical Detection of Nucleic Acids". Nano Lett. 11, 3974-3978 (2011). Figure 1

MiR-488-3p Represses Malignant Behaviors and Facilitates Autophagy of Osteosarcoma Cells by Targeting Neurensin-2.

Osteosarcoma (OS) is a primary bone sarcoma that primarily affects children and adolescents and poses significant challenges in terms of treatment. microRNAs (miRNAs) have been implicated in OS cell growth and regulation. This study sought to investigate the role of hsa-miR-488-3p in autophagy and apoptosis of OS cells. The expression of miR-488-3p was examined in normal human osteoblasts and OS cell lines (U2OS, Saos2, and OS 99-1) using RT-qPCR. U2OS cells were transfected with miR-488- 3p-mimic, and cell viability, apoptosis, migration, and invasion were assessed using CCK-8, flow cytometry, and Transwell assays, respectively. Western blotting and immunofluorescence were employed to measure apoptosis- and autophagy-related protein levels, as well as the autophagosome marker LC3. The binding sites between miR-488-3p and neurensin-2 (NRSN2) were predicted using online bioinformatics tools and confirmed by a dual-luciferase assay. Functional rescue experiments were conducted by co-transfecting miR-488-3p-mimic and pcDNA3.1-NRSN2 into U2OS cells to validate the effects of the miR-488-3p/NRSN2 axis on OS cell behaviors. Additionally, 3-MA, an autophagy inhibitor, was used to investigate the relationship between miR- 488-3p/NRSN2 and cell apoptosis and autophagy. miR-488-3p was found to be downregulated in OS cell lines, and its over-expression inhibited the viability, migration, and invasion while promoting apoptosis of U2OS cells. NRSN2 was identified as a direct target of miR-488-3p. Over-expression of NRSN2 partially counteracted the inhibitory effects of miR-488-3p on malignant behaviors of U2OS cells. Furthermore, miR- 488-3p induced autophagy in U2OS cells through NRSN2-mediated mechanisms. The autophagy inhibitor 3-MA partially reversed the effects of the miR-488-3p/NRSN2 axis in U2OS cells. Our findings demonstrate that miR-488-3p suppresses malignant behaviors and promotes autophagy in OS cells by targeting NRSN2. This study provides insights into the role of miR-488-3p in OS pathogenesis and suggests its potential as a therapeutic target for OS treatment.