PMCID string | Title string | Sentences string |
|---|---|---|
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Nonetheless, we have previously reported a similar extracellularly membrane-bound ChAT in human spermatozoa . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | There are reports indicating the presence of intracellularly membrane-bound ChAT in synaptic vesicles and synaptosomes across several animal species . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Intriguingly, ACh synthesized by membrane-bound ChAT appears to be approximately 40% more resistant to hydrolysis by AChE than ACh produced by the soluble form of ChAT . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In a recent study, we also demonstrated that human ChAT has a strong propensity to associate with membrane-like micelles, a phenomenon that enhances its enzymatic activity by more than tenfold . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Therefore, the distinct extracellular localization of ChAT in the SH-SY5Y neuroblastoma cell line may represent a novel physiologically relevant cholinergic-enhancing mechanism, potentially involving rapid in situ synaptic recycling of ACh and prolonging its action at the synapse. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This interpretation aligns with our previous hypothesis regarding extracellularly membrane-bound ChAT in human spermatozoa, where in situ ACh synthesis was proposed to occur in close proximity to membrane-bound receptors, enabling timely chemotactic signaling to facilitate or regulate sperm motility . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | For comparison, we also examined ChAT expression in two SCLC neuroendocrine cell lines (H82 and H69) and one NSCLC lung adenocarcinoma cell line (A549). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Although these cells lacked detectable extracellularly membrane-bound ChAT, they exhibited intracellular ChAT staining comparable to that observed in SH-SY5Y cells. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | While only four cancer cell lines were analyzed, our findings suggest that extracellular ChAT localization is not a general feature of cancer cells. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | To assess whether ChAT expression in these cell lines has functional relevance, we measured ACh production and release. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Despite differences in synthesis and release levels among the cell lines, all four were capable of synthesizing and releasing ACh. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | These findings are consistent with previous reports on ACh and choline production in human colon cancer cell cultures , as well as in SCLC, where the H82 cell line—similar to our study—exhibited higher ACh levels than H69 cells . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The cholinergic machinery includes, among other components, various forms of nicotinic and muscarinic ACh receptors (nAChRs and mAChRs), as well as ACh-degrading enzymes such as AChE and BChE. Many of these components have previously been reported in SCLCs and in the neuroblastoma SH-SY5Y cell line . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | We therefore examined several of these components in our study. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In SH-SY5Y cells, we first analyzed the presence of surface-localized AChE, BChE, M1-mAChR, and α7-nAChR using both flow cytometry and confocal microscopy. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Both M1-mAChR and α7-nAChR were detected extracellularly, which is expected given their known integration into the plasma membrane and previous reports of their presence in various cancer cells . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The observation of extracellularly membrane-bound BChE, rather than AChE, is particularly noteworthy. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | If confirmed by further experiments, this finding would challenge the prevailing view that AChE is the primary membrane-anchored ACh-hydrolyzing enzyme found extracellularly in multimeric forms—such as on blood cells, at neuromuscular junctions, and within synaptic clefts of cholinergic interfaces in the brain . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | BChE, by contrast, has traditionally been considered a soluble enzyme. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | However, recent studies have suggested the existence of membrane-anchored AChE-BChE hybrid molecular forms . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | While our findings support this notion, the negligible or absent surface staining of AChE in SH-SY5Y cells suggests that membrane-anchored BChE is the dominant form in this cell line. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Upon membrane permeabilization, flow cytometric analyses reflect antibody interaction with both surface and intracellular protein pools. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | As shown in Figure 6B, a comparison of relative fluorescence intensity between total and surface AChE and BChE signals in SH-SY5Y cells suggested that BChE was significantly more abundant than AChE, supporting the conclusion that BChE is the predominant membrane-anchored cholinesterase. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Confocal microscopy further corroborated this (Figure 7 and Supplementary 3D-Video S3 and S4 vs. S5 and S6), showing an even distribution of BChE across the cell surface, whereas AChE displayed a punctate, localized pattern suggestive of discrete focal expression. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Nonetheless, this conclusion should be interpreted with caution, as different antibodies targeting distinct proteins were used, and variations in antibody affinity may influence staining intensity. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Furthermore, we investigated the expression of cholinergic signaling biomolecules in lung cancer cell lines, specifically the SCLCs H82 and H69, as well as the lung adenocarcinoma cell line A549. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The results closely resembled those observed in the neuroblastoma SH-SY5Y cell line, not only in terms of ChAT expression and ACh synthesis and release, but also regarding the expression patterns of AChE, BChE, M1-mAChR, and α7-nAChR. Notably, in all three cell lines, the total staining intensity of AChE appeared significantly lower than that of BChE (Figure 6B, also see the Supplementary 3D Video S3–S6). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Interestingly, previous reports have described hybrid BChE-AChE molecular forms in human glioma . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Given that the examined cell lines are of cancerous origin, this phenomenon may represent a distinct feature of cancer pathology. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | For example, BChE is known to possess scavenging properties against cytotoxic agents naturally ingested through sources such as food. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Thus, elevated BChE expression may provide tumor cells with a protective mechanism against such agents. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Indeed, one study reported increased BChE levels in the serum of patients with cancers of various tissue origins following treatment, compared to baseline levels or healthy controls .Our findings suggest that screening current cytotoxic agents for their susceptibility to BChE metabolism is warranted, to identify compounds that are not metabolized by BChE and may therefore be more effective under these conditions. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Moreover, the presence of key components of the cholinergic system—including its central enzyme, ChAT—raises the possibility of a significant role for cholinergic signaling in cancer cell biology. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | However, based on our current data, the presence or expression of cholinergic components should not be interpreted as causative of cancer, as no normal human cell lines were included for comparison. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Nevertheless, our findings align with existing reports indicating that cholinergic signaling promotes cell survival and proliferation in various cancers, including lung cancer . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Therefore, further research into the role of cholinergic signaling in cancer biology is imperative, as it may contribute to the development of tolerance to therapeutic insults such as chemo- and/or radiotherapy. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Inhibiting this pathway could potentially enhance the efficacy of such treatments. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | We have previously hypothesized that ACh may also function as an intracellular signaling molecule involved in monitoring mitochondrial bioenergetics and regulating mitochondria-driven apoptosis , a process that may be differentially altered in cancer and neurodegenerative diseases such as AD and ALS. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | According to our hypothesis, intracellular ACh plays a critical role in protecting cells from mitochondria-driven apoptosis (Figure 9). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This could help explain how two seemingly distinct disorders may share a common pathological feature. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In neurodegenerative diseases (e.g., AD and ALS), selective degeneration occurs in central cholinergic neurons, spinal and peripheral cholinergic motor neurons, and parasympathetic cranial nerves, accompanied by a marked reduction in ChAT expression . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Consequently, the expected decline in intracellular ACh biosynthesis via ChAT may reduce cellular protection against mitochondrial apoptosis, thereby facilitating neuronal degeneration. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In contrast, tumor cells appear to upregulate a pronounced cholinergic phenotype to shield themselves from apoptosis . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Maintaining continuous intracellular ACh biosynthesis for such protective purposes is a highly energy-demanding process, as it requires equimolar amounts of acetyl-CoA—normally used for ATP production. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This suggests that upregulated ACh signaling in tumor cells may represent a strategic energy investment, especially considering the high energy demands of cancer cell proliferation. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Thus, the acetylcholine–mitochondria–apoptosis axis emerges as a critical area of investigation for understanding both selective cholinergic neurodegeneration in dementia and neuromotor disorders, and the survival strategies employed by tumor cells, including neuroblastoma and lung cancer. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | An important question that remains unresolved in the current study is whether ChAT is fully localized extracellularly—similar to how AChE is anchored to the membrane via specific anchoring proteins ,—or whether it is partially embedded within the plasma membrane, as previously demonstrated in micellar particles , synaptic vesicles, and synaptosomes . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In our flow cytometric analysis, we used an anti-ChAT antibody (Abcam Cat# ab181023, RRID: AB_2687983) that recognizes an epitope near the C-terminal domain of the ChAT protein (around amino acid 700). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This suggests that the C-terminal region of ChAT is exposed on the extracellular surface of the plasma membrane. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | To further investigate ChAT localization, we employed three additional antibodies in confocal analyses. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | One was a monoclonal mouse anti-ChAT antibody (MAB3447, R&D Systems), which targets a region between Ala2 and Pro630 (Accession # NP_066266). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | However, this epitope range does not allow us to determine whether the antibody binds to the N-terminus, C-terminus, or an internal domain of the protein. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The other two antibodies—rabbit polyclonal anti-ChAT antibodies from Abnova (PAB14536) and Millipore (AB143)—likely contain subclones that recognize multiple epitopes across the ChAT sequence, further complicating precise localization. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Nonetheless, our findings suggest that ChAT is either embedded in the plasma membrane or fully extracellular but anchored to the membrane, as it was not washed away during surface staining procedures. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This contrasts with our previous observations of sperm surface ChAT, which was readily removed under similar conditions . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The retention of ChAT on the cell surface implies a stable membrane association, but the nature of this interaction—whether via transmembrane domains, lipid anchors, or protein–protein interactions—remains to be elucidated. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Taken together, while our data strongly support the presence of extracellularly exposed ChAT, the exact membrane topology and anchoring mechanism remain uncertain. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Addressing this question will require additional studies, including epitope mapping, protease protection assays, and advanced imaging techniques such as cryo-electron microscopy or proximity labeling. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Clarifying the localization and membrane association of ChAT is essential for understanding the mechanism of in situ ACh signaling and its potential role in cancer cell survival, communication, and therapeutic targeting. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In summary, this study provides compelling evidence for the presence of an extracellular, membrane-bound form of ChAT exclusively in neuroblastoma cells, in contrast to the three lung cancer cell lines tested. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | However, all examined cell lines expressed intracellular ChAT. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Additionally, all cells expressed several other components of the cholinergic machinery, reinforcing the notion that ChAT expression serves a biological function consistent with ACh signaling. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | These findings highlight the complexity of cholinergic signaling, particularly the distinct roles of membrane-bound and intracellular cholinergic markers. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Dysregulation of these components has been linked to neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Membrane-bound and intracellular cholinergic markers influence ACh homeostasis, receptor trafficking, apoptotic pathways, and neuroinflammation, making them critical targets for therapeutic intervention. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Further research is needed to elucidate the molecular pathways regulating these markers and to assess their potential as therapeutic targets in both neurodegenerative diseases and cancer. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | A deeper understanding of their interactions and regulatory mechanisms could pave the way for strategies aimed at restoring cholinergic system integrity and mitigating disease progression. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This study employed a range of reagents and antibodies essential for cell culture and flow cytometric analysis. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | RPMI-1640 medium, fetal bovine serum (FBS), trypsin, penicillin, and streptomycin were sourced from Thermo Fisher Scientific (Stockholm, Sweden), along with fixation and permeabilization buffers (Cat# 00-8333-56). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Cell culture containers, including T25 and T75 flasks, plates, and 15 mL Falcon tubes, were obtained from Corning (via Merck, Darmstadt, Germany). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | For the detection of ChAT via flow cytometry, an APC-conjugated recombinant anti-ChAT antibody (Cat# AB224001, Abcam (Cambridge, UK)) was used, which targets a sequence at amino acid 700 [https://www.antibodyregistry.org/AB_2687983 (accessed on 16 October 2025)]. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Additional cholinergic markers were analyzed using FITC-conjugated anti-AChE antibody (A-11, Sc-373901), PE-conjugated anti-BChE antibody (D-5, Sc-377403), AF647-conjugated anti-α7 AChR antibody (319, Sc-58607), and PE-conjugated anti M1 AChR antibody (G-9, Sc-365966), all from Santa Cruz Biotechnology (Dallas, TX, USA). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Surface staining was performed without fixation, using a 1:1500 dilution of LIVE/DEAD™ Cell Stain dye (Cat# L23101, Invitrogen, Thermo Fisher Scientific (Stockholm, Sweden)) to assess membrane integrity, as previously described . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | For intracellular staining, cells were treated with permeabilization buffer (Cat# 88-8824-00, eBioscience™, via Thermo Fisher Scientific) following the manufacturer’s protocol. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Recombinant human ChAT protein (5.1 mg/mL) was produced and purified by the Protein Science Facility at Karolinska Institutet (Stockholm, Sweden), as previously reported . |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | The study utilized four human cell lines: two small cell lung carcinoma lines (NCI-H69 and NCI-H82), one lung adenocarcinoma line (A549), and one neuroblastoma line (SH-SY5Y), all obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin, and maintained at 37 °C in a humidified incubator with 5% CO2. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Adherent cell lines (A549 and SH-SY5Y) were detached using 0.25% trypsin and 0.02% EDTA once they reached 80–90% confluency. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In contrast, the non-adherent SCLC lines (H69 and H82) were maintained in suspension and processed by centrifugation and washing without enzymatic detachment. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Cell Line Selection Rationale: We selected the SH-SY5Y neuroblastoma cell line due to its extensive characterization in the literature and the availability of well-established differentiation protocols , which have been successfully implemented in our laboratory. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | This cell line will be utilized in upstream studies investigating ChAT ligands as potential therapeutics for neurodegenerative disorders such as AD and ALS. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Additionally, the small-cell lung cancer cell line (H69 and H82) was chosen because disease relapses following an initially favorable response to chemotherapy remains a significant clinical challenge. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Despite ongoing research, few novel therapeutic targets have demonstrated meaningful clinical efficacy , underscoring the urgent need for innovative approaches. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Flow cytometric analysis was conducted to assess the expression of cholinergic markers in both adherent and suspension cell lines. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | For A549 and SH-SY5Y cells, staining was performed after detachment, washing in PBS, and cell counting. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Suspension cells (H69 and H82) were directly centrifuged, washed, and counted. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Approximately 1 × 10 cells were used for each staining condition. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Surface staining involved simultaneous incubation of cells with LIVE/DEAD™ dye and the respective conjugated antibody in PBS containing 0.5% BSA, for 30 min at 4 °C. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | These cells were not fixed or permeabilized. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Whole-cell staining was performed following fixation in 4% paraformaldehyde for 10 min at room temperature, washing, and permeabilization according to the manufacturer’s instructions. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Cells were then incubated with primary antibodies at specified dilutions for 30 min at 4 °C, washed, and resuspended in PBS for analysis using a CytoFLEX S flow cytometer (Beckman Coulter, Stockholm, Sweden). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | To validate the specificity of ChAT antibody staining, cells were incubated with recombinant ChAT protein (~102 μg/mL) alongside the anti-ChAT antibody (1:1250 dilution) before staining. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | Similar protocols were applied for AChE, BChE, α7-AChR, and M1-AChR, using a 1:50 dilution for each antibody. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | All experiments were performed in triplicate. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In selected experiments, protein expression was quantified as relative fluorescence intensity (RFI), calculated by dividing the geometric mean fluorescence intensity (GM-FI) of the marker by that of its control, using FlowJo software (v10.10.0). |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | For ChAT, the control consisted of cells stained with the blocked antibody, while for other markers, cells stained only with LIVE/DEAD dye served as controls. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | To compare total versus surface expression, RFI was calculated by dividing GM-FI from whole-cell staining by GM-FI from surface staining of the same marker. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | To assess acetylcholine (ACh) levels, cells were cultured in 12-well plates for 24 to 48 h. For non-adherent cell lines (H69 and H82), the entire contents of the wells were transferred to Falcon tubes and centrifuged at 3000 rpm. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | From these, 1–2 mL of the supernatant—representing the conditioned medium—was collected for subsequent ACh and choline measurements. |
PMC12608971 | Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells | In the case of adherent cell lines (SH-SY5Y and A549), the conditioned medium was collected directly using a pipette and transferred to Eppendorf tubes. |
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