ANALYTICAL CHEMISTRY
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2023
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IF 7.4
Li Xu,Haiyang Xie,Boqian Wang,Zijian Zhu,Hui Jiang,Xiaoqian Duan,Shuxin Deng,Jiasu Xu,Lai Jiang,Xianting Ding
Single-cell western blotting (scWB) is a prevalent technique for high-resolution protein analysis on low-abundance cell samples. However, the extensive signal loss during repeated antibody stripping precludes multiplex protein detection. Herein, we introduce Fluorescent-quenching Aptamer-based Single-cell Western Blotting (FAS-WB) for multiplex protein detection at single-cell resolution. The minimal size of aptamer probes allows rapid in-gel penetration, diffusion, and elution. Meanwhile, the fluorophore-tagged aptamers, coordinated with complementary quenching strands, avoid the massive signal loss conventionally caused by antibody stripping during repeated staining. Such a strategy also facilitates multiplex protein analysis with a limited number of fluorescent tags. We demonstrated FAS-WB for co-imaging four biomarker proteins (EpCAM, PTK7, HER2, CA125) at single-cell resolution with lower signal loss and enhanced signal-to-noise ratio compared to conventional antibody-based scWB. Being more time-saving (less than 25 min per cycle) and economical (1/1000 cost of conventional antibody probes), FAS-WB offers a highly efficient platform for profiling multiplex proteins at single-cell resolution.
研究领域:
生物化学
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分子生物学
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医学诊断
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
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2024
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IF 6.7
Xiaojun Li,Xiulei Qi,Xingguo Liu,Jun Zhu,Lianghai Hu
Outer membrane vesicles (OMVs) secreted by bacteria are emerging diagnostic markers for bacterial infection or disease detection due to their carriage of various signaling molecules. However, actual biological samples of patients are extremely complex, and applying OMVs to clinical diagnosis remains a major challenge. One of the major challenges is that there are still great difficulties in the enrichment of OMVs including tedious steps and lower concentration. And some commonly used exosome enrichment methods, such as ultracentrifugation, still have some shortcomings. Herein, we introduce lipopolysaccharide (LPS) molecularly imprinted polymer (MIP) for efficient capturing and analyzing OMVs, enabling a novel approach to bacterial disease diagnosis based on biorecognition materials. LPS, as a unique structure of Gram-negative bacteria, also widely expressed on the surface of OMVs, which will form cyclic hydrogen bonds with functional monomers of MIP with affinity interactions. The prepared MIP efficiently can isolate OMVs from 100 μL of culture broth via specific affinity LPS in less than 40 min with a recovery rate of over 95%. Moreover, MIP exhibits good reusability, with almost identical enrichment performance after 5 repeated cycles, contributing to reducing experimental costs in both time and economy. The captured OMVs can be detected using Western blotting with target protein antibodies or in combination with proteomic analysis, providing a proteomic biomarker platform for early bacteria disease diagnosis.
研究领域:
生物材料
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生物技术
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微生物学
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诊断医学
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DOI
PubMed
高影响因子
MEDICAL SCIENCE MONITOR
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2020
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IF 1.918
Yan Dong,Jun Yang,Liyuan Yang,Ping Li
Background Quercetin (Que) is reported to induce apoptosis of lung cancer cells. Src is closely related to the progression of non-small cell lung cancer (NSCLC) and can be modulated by Que in macrophages. In the current study, the interaction between Que and Src signaling in NSCLC cells was explored to explain the anti-NSCLC function of Que. Material/Methods NSCLC cell line HCC827 was subjected to the administrations of Que at different concentrations. The effect of Que on tumor cell proliferation was detected using MTT and colony formation assays. Then the effect on the migration and invasion abilities was assessed using scratch and Transwell assays. At molecular level, the changes in Src/Fn14/NF-κB signaling were determined using western blotting assays. The role of Src in the function of Que was further explored by inducing the expression of Src gene in NSCLC cells before Que administration. The results of the in vitro assays were verified using a NSCLC mice model. Results Que inhibited the proliferation and anchorage-independent growth of NSCLC cells. Additionally, Que delayed in the gap closure rate in scratch assays and decreased the membrane-penetrating cell number in Transwell assays. At a molecular level, Que suppressed the expression of Src, which subsequently inhibited Fn14/NF-κB signaling. In in vivo assays, Que inhibited the growth of solid tumors. After the overexpression of Src in NSCLC cells, the anti-NSCLC effect of Que was blocked by inducing NSCLC proliferation and metastasis, and by activating Fn14/NF-κB signaling. Moreover, the induced level of Src promoted the growth and metastasis potential of solid tumors in mice. Conclusions Que exerted the anti-NSCLC effect by inhibiting Src-mediated Fn14/NF-κB pathway both in vitro and in vivo .
研究领域:
肿瘤学
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药理学
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细胞生物学
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分子生物学
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DOI
PubMed
高影响因子
Frontiers in Pharmacology
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2024
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IF 4.4
Feng Sa,Liu Linyan,Cheng Yuelin,Zhou Mengmeng,Zhu Haoqiang,Zhao Xinyan,Chen Ziyu,Kan Shunli,Fu Xuanhao,Hu Wei,Zhu Rusen
Objective: The limited ability to regenerate axons after spinal cord injury (SCI) is influenced by factors such as astrocyte activation, reactive proliferation, and glial scar formation. The TGF-β/Smad (transforming growth factor-β/mothers against decapentaplegic homolog) pathway, associated with astrocytic scarring, plays a crucial role in recovery post-injury. This study aims to investigate how icariin (ICA) interacts with reactive astrocytes in the treatment of spinal cord injury.Methods: A rat SCI model was constructed, and the recovery of motor function was observed after treatment with ICA.HE staining, LFB staining, immunofluorescence staining, and Western blotting were employed to assess ICA's ability to inhibit astrocyte proliferation in rats following spinal cord injury by modulating YAP, as well as to evaluate the reparative effects of ICA on the injured spinal cord tissue. Primary astrocytes were isolated and cultured. Immunoprecipitation-Western Blot (IP-WB) ubiquitination and cytoplasm-nuclear separation were employed to assess PPM1B ubiquitination and nuclear translocation.Results: The CatWalk XT gait analysis, BBB (Basso, Beattie, and Bresnahan) score, electrophysiological measurements, HE staining, and LFB staining collectively demonstrated that ICA promotes motor function and tissue recovery following spinal cord injury in rats. Immunofluorescence staining and Western Blot analyses revealed that ICA inhibits astrocyte proliferation in rats post-spinal cord injury by suppressing YAP activity. Furthermore, the activation of YAP by XMU-MP-1 was shown to compromise the efficacy of ICA in these rats after spinal cord injury. Additional immunofluorescence staining and Western Blot experiments confirmed that ICA inhibits TGFβ1-induced astrocyte activation through the regulation of YAP. The knockdown of PPM1B (protein phosphatase, Mg2+/Mn2+-dependent 1B) in astrocytes was found to inhibit TGFβ signaling. Additionally, YAP was shown to regulate PPM1B ubiquitination and nuclear translocation through immunoprecipitation-Western blot analysis, along with the segregation of cytoplasm and nucleus.Conclusion: Icariin promotes functional recovery in rats after spinal cord injury by inhibiting YAP and regulating PPM1B ubiquitination to inhibiting the activation of reactive astrocytes.
研究领域:
神经科学
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药理学
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分子生物学
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细胞生物学
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产品关联
DOI
PubMed
高影响因子
TOXICOLOGY
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2023
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IF 4.5
Yuting Gao,Wenli Liao,Ruyi Zhang,Yining Lei,Tao Chen,Lingling Wu,Manqin Li,Xinran Liu,Fei Cai
Neuroinflammation is one of the important mechanisms of trimethyltin chloride (TMT) central neurotoxicity . Artemisinin (ARS) is a well-known antimalarial drug that also has significant anti-inflammatory effects. Prokineticin 2 (PK2) is a small molecule secreted protein that is widely expressed in the nervous system and plays a key role in the development of neuroinflammation. However, it remains unclear whether ARS can ameliorate neuroinflammation caused by TMT and whether PK2/PKRs signaling pathway plays a part in it. In this research, male Balb/c mice were administered TMT (2.8 mg/kg, i.p.) followed by immunohistochemistry to assess the expression of PK2, PKR1, and PKR2 proteins in the hippocampus. Network pharmacology was used to predict the intersection targets of ARS, central nervous system(CNS) injury and TMT. The neurobehavior of mice was evaluated by behavioral scores. Histopathological damage of the hippocampus was evaluated by HE, Nissl and Electron microscopy . Western blotting was used to identify the expression of synapse-related proteins (PSD95, SYN1, Synaptophysin), PK system-related proteins (PK2, PKR1, PKR2), and inflammation-related proteins (TNF-α, NF-κB p65). Immunohistochemistry showed that TMT resulted in elevated PK2 and PKR2 protein expression in the CA2 and CA3 regions of the hippocampus in mice, while PKR1 protein was not significantly altered. Network pharmacology showed that PK2 could interact with the intersectional targets of ARS, CNS injury, and TMT. ARS remarkably attenuated TMT-induced seizures and hippocampal histological damage. Further studies demonstrated that ARS treatment attenuated TMT-induced hippocampal ultrastructural damage, possibly by increasing the number of rough endoplasmic reticulum and mitochondria as well as upregulating the levels of synapse-associated proteins (PSD95, SYN1, Synaptophysin). Western blotting results revealed that ARS downregulated TMT-induced TNF-α and NF-κB p65 protein levels. In addition, ARS also decreased TMT-induced protein expression of PK2 and PKR2 in the mouse hippocampus, but had no significant effect on PKR1 protein expression. Our results suggested that ARS ameliorated TMT-induced abnormal neural behavior and hippocampal injury, which may be achieved by regulating PK2/PKRs inflammatory pathway and ameliorating synaptic injury. Therefore, we suggest that PK2/PKRs pathway may be involved in TMT neurotoxicity and ARS may be a promising drug that can relieve TMT neurotoxicity.
研究领域:
神经科学
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药理学
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毒理学
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产品关联
DOI
PubMed
高影响因子
Cancer Cell International
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2021
·Vol. 21
·期 1
·1-12
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IF 5.722
Jiang Dongxiao,Ding Shufei,Mao Zhujun,You Liyan,Ruan Yeping
Background Colon cancer is a malignant gastrointestinal tumour with high incidence, mortality and metastasis rates worldwide. Aloe-emodin is a monomer compound derived from hydroxyanthraquinone. Aloe-emodin produces a wide range of antitumour effects and is produced by rhubarb, aloe and other herbs. However, the mechanism by which aloe-emodin influences colon cancer is still unclear. We hope these findings will lead to the development of a new therapeutic strategy for the treatment of colon cancer in the clinic.Methods We identified the overlapping targets of aloe-emodin and colon cancer and performed protein–protein interaction (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. In addition, we selected apoptosis pathways for experimental verification with cell viability, cell proliferation, caspase-3 activity, DAPI staining, cell cycle and western blotting analyses to evaluate the apoptotic effect of aloe-emodin on colon cancer cells.Results The MTT assay and cell colony formation assay showed that aloe-emodin inhibited cell proliferation. DAPI staining confirmed that aloe-emodin induced apoptosis. Aloe-emodin upregulated the protein level of Bax and decreased the expression of Bcl-2, which activates caspase-3 and caspase-9. Furthermore, the protein expression level of cytochrome C increased in a time-dependent manner in the cytoplasm but decreased in a time-dependent manner in the mitochondria.Conclusion These results indicate that aloe-emodin may induce the apoptosis of human colon cancer cells through mitochondria-related pathways.
研究领域:
肿瘤学
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药理学
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分子生物学
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细胞生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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