STAR Protocols
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2023
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IF 0
Jiaqi Shi,Caiqi Liu,Shengnan Luo,Yingying Zhang,Mingwei Li,Meng Zhou,Mingjiao Weng,Xiaobo Li,Tongsen Zheng
Summary Here, we present a protocol for the detection of the two STING isoforms (erSTING and pmSTING) in human peripheral blood mononuclear cells or mouse splenocytes using Western blot and PCR. We detail steps to construct plasmids encoding each isoform and transfer them into mouse and human cell lines. Finally, we describe how to detect cell membrane localization of pmSTING using flow cytometry, immunoprecipitation, and immunofluorescence. This protocol is applicable for proteins with well-predicted topological structures. For complete details on the use and execution of this protocol, please refer to Li et al. 1 .
研究领域:
分子生物学
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细胞生物学
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免疫学
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DOI
PubMed
Plants-Basel
·
2025
·Vol. 14
·期 22
·
IF 4.1
Dong Li,Xumin Wang,Yujiao Song,Jianchao Sun,Shuhan Yu,Bowei Zhu,Xin Liu,Guodong Zhao,Tongsheng Zhao,Limin Wang
Seed oil content and fatty acid (FA) composition collectively determine the quality and economic value ofBrassica napus. Little is known about the role of R2R3-MYB transcription factors (TFs) in regulating FA biosynthesis inB. napus. Here,BnaC04.MYB89was found to be expressed primarily in developing seeds. Overexpression ofBnaC04.MYB89consistently decreased FA levels, as evidenced by its effect in both theArabidopsis thaliana myb89-1mutant andB. napusseeds. RNA-seq of developing seeds at 30 DAP (days after pollination) revealed marked suppression of FA biosynthetic genes inBnaC04.MYB89-overexpressing plants compared to the K407 control. ChIP (Chromatin immunoprecipitation) analysis revealed that BnaC04.MYB89 directly inhibited the expression ofBnaA03.BCCP1andBnaC03.HDwhile indirectly regulating that ofBnaA09.BADC1,BnaA03.BADC3,BnaA03.MOD1, andBnaA08.FAT8, thereby reducing seed FA accumulation. Collectively, these results elucidate the role for BnaC04.MYB89 and provide new insights into the transcriptional regulatory network controlling seed oil accumulation inB. napus.
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DOI
PubMed
高影响因子
Frontiers in Genetics
·
2021
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IF 4.599
Li Jia,Shen Jie,Qin Lan,Lu Dongyan,Ding Enci
Background: Thyroid cancer is a frequent endocrine tumor in women. It is of great significance to investigate the molecular mechanism of progression of thyroid cancer.Methods: Gene expression data set and clinical data were downloaded from The Cancer Genome Atlas database for differential expression analysis. The triplet of downstream transcription factors (TFs) and modulatory genes of target lncRNA in thyroid cancer was predicted by the lncMAP database. mRNA and protein expression of lncRNA LBX2-AS1, RARα, and FSTL3 were detected by qRT-PCR and western blot. The localization of lncRNA LBX2-AS1 in cells was tested by Fluorescence in situ hybridization assay. The RNA immunoprecipitation assay was applied to verify the binding relationship between lncRNA LBX2-AS1 and FSTL3. ChIP and dual-luciferase assays were used to prove the binding relationship between RARα and FSTL3. Cell function experiments were used to test cell proliferation, migration and invasion in each treatment group. The role of lncRNA LBX2-AS1 in thyroid cancer progression was also confirmed in nude mice.Results: Bioinformatics analysis indicated that lncRNA LBX2-AS1, RARα, FSTL3 were remarkably fostered in thyroid cancer tissue, and LBX2-AS1 was evidently correlated with clinical features. The LncMAP triplet prediction showed that LBX2-AS1 recruited TF RARα to modulate FSTL3. RIP assay confirmed that LBX2-AS1 was prominently enriched on RARα. ChIP and dual-luciferase report assays unveiled that RARα bound to the promoter region of FSTL3 and functioned as a TF. Cell function experiments uncovered that LBX2-AS1 boosted the progression of thyroid cancer. The rescue experiments showed that LBX2-AS1 recruited the TF RARα to hasten the transcription activity of FSTL3 and thus promoted the development of thyroid cancer.Conclusion: The integrative results demonstrated that LBX2-AS1 activated FSTL3 by binding to TF RARα to hasten proliferation, migration and invasion of thyroid cancer.
研究领域:
肿瘤学
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分子生物学
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癌症遗传学
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生物信息学
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产品关联
DOI
PubMed
高影响因子
SCIENTIA HORTICULTURAE
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2024
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IF 3.9
Qiong Wu,Xiaoya Tao,Chunxiao Cui,Yanan He,Dongdong Zhang,Yurong Zhang,Li Li
Phytohormones are vital for tomato ripening, and abscisic acid (ABA) is speculated to induce ethylene biosynthesis at the initiate of tomato fruit ripening; however, its interaction sites remain unclear. In the present study, Constans-like 4 (SlCOL4), a CO/COL transcription factor, was cloned from tomato fruit. Amino acid sequence and subcellular location analysis proved that SlCOL4 was a typical transcription factor of the COL family. Gene expression analysis showed SlCOL4 expressed in different tissues of tomato plants and its transcription abundance declined at the onset of fruit ripening, coincidentally it was found to be down-regulated in ABA-treated tomato fruit and responded to exogenous ABA within 6 h after treatment. Meanwhile, yeast one-hybrid and dual-luciferase analysis showed that the ABA response factor SlAREB1 inhibited the transcription of SlCOL4 , indicating it was directly regulated by ABA at the mature green stage of tomato fruit . In addition, the pericarp color turned red faster than that of control fruit, and the key ethylene biosynthesis genes, ACS2, ACS4, and ACO1 , were up-regulated in SlCOL4 silenced tomato fruit, suggesting that SlCOL4 may be a negative regulator of ripening. Furthermore, several genes associated with the ethylene signaling pathway were identified downstream of SlCOL4 by chromatin immunoprecipitation sequencing (ChIP-Seq) analysis. Overall, the transcription factor SlCOL4 may interact with ABA and ethylene signals and negatively regulate tomato fruit ripening. The results in this study provide new insights for elucidating the molecular mechanism of ABA-ethylene interaction in regulating tomato fruit ripening, and provide references for further research on the role of COLs transcription factors in tomato fruit ripening as well.
研究领域:
植物生理学
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分子生物学
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园艺学
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DOI
高影响因子
MOLECULES
·
2022
·Vol. 27
·期 14
·4556
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IF 4.927
Xin-Yi Tong,Xin-Zhi Yang,Shu-Qin Gao,Xiao-Juan Wang,Ge-Bo Wen,Ying-Wu Lin
Imbalance in the cellular redox system is thought to be associated with the induction and progression of breast cancers, and heme proteins may regulate the redox balance. Cytochromeb5(Cytb5) is a small mitochondrial heme protein. Its function and regulating mechanism in breast cancer remain unknown. In this study, we elucidated the level of endogenous oxidative stress in breast cancer cells, MCF-7 cells (hormone receptor-positive cells) and MDA-MB-231 cells (triple-negative cells), and investigated the difference in Cytb5content. Based on the low content of Cytb5in MDA-MB-231 cells, the overexpression of Cytb5was found to regulate the oxidative stress and apoptosis cascades, including ERK1/2 and Akt signaling pathways. The overexpressed Cytb5MDA-MB-231 cells were shown to exhibit decreased oxidative stress, less phosphorylation of ERK1/2 and Akt, and less cleavage of caspases 3 and 9 upon treatment with H2O2, as compared to those of normal MDA-MB-231 cells. Moreover, the overexpressed Cytb5most likely functioned by interacting with its protein partner, Cytc, as suggested by co-immunoprecipitation studies. These results indicated that Cytb5has different effects on breast cancer cells of different phenotypes, which provides useful information for understanding the multiple roles of Cytb5and provides clues for clinical treatment.Keywords:Cytochromeb5;Cytochromec;protein-protein interactions;breast cancer cells;co-immunoprecipitation
研究领域:
癌症生物学
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细胞生物化学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
Journal of Proteomics
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2016
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IF 3.867
Wen Zhang,Ting Zhong,Yun Chen
In breast cancer, p53 could be functionally compromised by interaction with several proteins. Among those proteins, MDM2 serves as a pivotal negative regulator and counteracts p53 activation. Thus, the ability to quantitatively and accurately monitor the changes in level of p53-MDM2 interaction with disease state can enable an improved understanding of this protein-protein interaction (PPI), provide a better insight into cancer development and allow the emergence of advanced treatments. However, rare studies have evaluated the quantitative extent of PPI including p53-MDM2 interaction so far. In this study, a LC-MS/MS-based targeted proteomics assay was developed and coupled with co-immunoprecipitation (Co-IP) for the quantification of p53-MDM2 complex. A p53 antibody with the epitope residing at 156–214 residues achieved the greatest IP efficiency. 321KPLDGEYFTLQIR333 (p53) and 327ENWLPEDK334 (MDM2) were selected as surrogate peptides in the targeted analysis. Stable isotope-labeled synthetic peptides were used as internal standards. An LOQ (limit of quantification) of 2 ng/mL was obtained. Then, the assay was applied to quantitatively detect total p53, total MDM2 and p53-MDM2 in breast cells and tissue samples. Western blotting was performed for a comparison. Finally, a quantitative time-course analysis in MCF-7 cells with the treatment of nutlin-3 as a PPI inhibitor was also monitored. Biological significance Proteins do not function as single entities but rather as a team player that has to communicate. Protein-protein interaction (PPI), normally by means of non-covalent contact among binary or large protein complex , is essential for many cellular processes including cancer progression. Thus, the ability to quantitatively and accurately monitor the changes in level of PPI with disease state can enable an improved understanding of PPI, provide a better insight into cancer development and allow the emergence of advanced treatments. However, rare studies have evaluated the quantitative extent of PPI so far. The major issue of current available approaches is the trade-off between sensitivity and specificity. Thus, techniques with the ability to quantify PPIs with both high sensitivity (low false-negative rate) and high specificity (low false-positive rate) are eagerly desired. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics has shown its potential to study biomolecules because of its high sensitivity, high selectivity and wide dynamic range. In this study, we made an effort to develop a LC-MS/MS-based targeted proteomics assay for the quantitative detection of p53-MDM2 interaction in breast cells and tissue samples.
研究领域:
癌症生物学
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蛋白质组学
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质谱分析
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分子生物学
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产品关联
DOI
PubMed
高影响因子
CHEMICO-BIOLOGICAL INTERACTIONS
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2024
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IF 4.7
Meng Luan,Bijun Zhang,Yifan Wei,Fanghua Liu,Yuhong Zhao,Yalian Yu,Qijun Wu
Enhanced drug resistance poses a significant challenge in treating ovarian cancer (OC). Phenylethyl isothiocyanate (PEITC) is involved in drug resistance in OC, but the mechanism remains unclear. In this study, we investigated the molecular regulatory mechanism of carboplatin sensitivity in OC associated with PEITC, MAF BZIP Transcription Factor F (MAFF), and Zinc finger proteins (ZNF) 711. The carboplatin sensitivity was significantly increased in OC cells after PEITC treatment. Knockdown of MAFF significantly enhanced the carboplatin sensitivity of OC cells, promoted apoptosis, inhibited colony-forming efficiency in vitro , and suppressed tumor growth in vivo . The binding site of MAFF to the ZNF711 promoter was predicted, and the knockdown of MAFF significantly increased the ZNF711 expression. Results of the dual luciferase assay and ChIP-PCR confirmed the binding of MAFF to the ZNF711 promoter. Immunofluorescence and CoIP results demonstrated the colocalization and the binding of MAFF and its interacting protein, BZIP Transcription Factor ATF-like 3 (BATF3). Similarly, we confirmed the binding of BATF3 to the ZNF711 promoter. Knockdown of BATF3 alone and simultaneous knockdown of BATF3 and MAFF showed similar regulatory effects on ZNF711 transcription and apoptosis. These suggested that the binding of MAFF to BATF3 inhibited ZNF711 transcription and reduced carboplatin sensitivity in OC.
研究领域:
肿瘤学
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分子生物学
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细胞生物学
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转化医学
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产品关联
DOI
PubMed
高影响因子
CANCER LETTERS
·
2023
·
IF 9.7
Tian-Jiao Li,Kai-Zhou Jin,Hong-Yu Zhou,Zhen-Yu Liao,Hui-Ru Zhang,Sai-Meng Shi,Meng-Xiong Lin,Shou-Jie Chai,Qing-Lin Fei,Long-Yun Ye,Xian-Jun Yu,Wei-Ding Wu
Pancreatic ductal adenocarcinoma (PDAC) is extremely malignant with limited treatment options. Deubiquitinases (DUBs), which cleave ubiquitin on substrates, can regulate tumor progression and are appealing therapeutic targets, but there are few related studies in PDAC. In our study, we screened the expression levels and prognostic value of USP family members based on published databases and selected USP10 as the potential interventional target in PDAC. IHC staining of the PDAC microarray revealed that USP10 expression was an adverse clinical feature of PDAC. USP10 promoted tumor growth both in vivo and in vitro in PDAC. Co-IP experiments revealed that USP10 directly interacts with PABPC1. Deubiquitination assays revealed that USP10 decreased the K27/29-linked ubiquitination level of the RRM2 domain of PABPC1. Deubiquitinated PABPC1 was able to couple more CLK2 mRNA and eIF4G1, which increased the translation efficiency. Replacing PABPC1 with a mutant that could not be ubiquitinated impaired USP10 knock-down-mediated tumor suppression in PDAC. Targeting USP10 significantly delayed the growth of cell-derived xenograft and patient-derived xenograft tumors. Collectively, our study first identified USP10 as the DUB of PABPC1 and provided a rationale for potential therapeutic options for PDAC with high USP10 expression.
研究领域:
肿瘤学
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分子生物学
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癌症生物学
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转化医学
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DOI
PubMed
高影响因子
STAR Protocols
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2022
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IF 0
Yajie Gao,Yuanxiang Zhu,Qinmiao Sun,Dahua Chen
Summary Here, we provide an optimized RNA-induced silencing complex (RISC) assembly and cleavage protocol in vitro without using radiolabeled RNA. The protocol is useful to characterize the biochemical properties of the RISC. We describe the preparation of RNA probes, the target RNA, and Drosophila cell lysates for RISC assembly assay. We then detail AGO1 complexes immunoprecipitation for RISC cleavage assay. This protocol can detect RISC assembly and cleavage products within 5 days. Moreover, it can detect 5′- and 3′-cleavage products simultaneously. For complete details on the use and execution of this protocol, please refer to Gao et al. (2022) .
研究领域:
分子生物学
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生物化学
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细胞生物学
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DOI
PubMed
Frontiers in Immunology
·
2023
·
IF 7.3
Zhang Lishan,Sun Lina,Srinivasan Ramanathan,Lin Meizhen,Gong Lanqing,Lin Xiangmin
Bacterial AraC is a transcription factor family that initiates transcription by recruiting RNA polymerase to the promoter and directly regulating various bacterial phenotypes. It also directly regulates various bacterial phenotypes. However, how this transcription factor regulates bacterial virulence and affects host immunity is still largely unknown. In this study, deleting the orf02889 (AraC-like transcription factor) gene in virulent Aeromonas hydrophila LP-2 affected several important phenotypes, such as increasing biofilm formation and siderophore production abilities. Moreover, Δorf02889 also significantly decreased the virulence of A. hydrophila and has promising attenuated vaccine potential. To better understand the effects of orf02889 on biological functions, a data independent acquisition (DIA)-based quantitative proteomics method was performed to compare the differentially expressed proteins between Δorf02889 and the wild-type strain in extracellular fractions. The following bioinformatics analysis suggested that ORF02889 may regulate various metabolic pathways, such as quorum sensing and ATP binding cassette (ABC) transporter metabolism. Moreover, 10 selected genes from the top 10 decreasing abundances in proteomics data were deleted, and their virulence to zebrafish was evaluated, respectively. The results showed that ΔcorC, Δorf00906, and Δorf04042 significantly reduced bacterial virulence. Finally, the following chromatin immunoprecipitation and polymerase chain reaction (ChIP-PCR) assay validated that the promoter of corC was directly regulated by ORF02889. Overall, these results provide insight into the biological function of ORF02889 and demonstrate its inherent regulatory mechanism for the virulence of A. hydrophila.
研究领域:
微生物学
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蛋白质组学
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分子生物学
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传染病学
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产品关联
DOI
PubMed
高影响因子
Biology Direct
·
2024
·Vol. 19
·期 1
·1-14
·
IF 5.7
Sun Tong,Yang Zhiguang
Six-transmembrane epithelial antigen of prostate 3 (STEAP3) is associated with the progression of several human malignancies. However, its role in lung squamous cell carcinoma (LUSC) remains unclear. We measured STEAP3 expression in LUSC cell lines and tissues. LUSC cells with stable STEAP3 overexpression and knockdown were obtained through G418 selection. Multiple assays were used to evaluate the malignant phenotypes of LUSC cells and the activation of the β-catenin signaling. The potential transcriptional regulatory factors of STEAP3 were predicted using the JASPAR database, and the correlation between transcription factor AP-2 gamma (TFAP2C) and STEAP3 was analyzed through the GEPIA database. The study evaluated the regulatory relationship between a potential transcription factor and STEAP3 through ChIP and luciferase reporter assays. Additionally, rescue assays were utilized to ascertain whether TFAP2C serves as the upstream regulatory factor of STEAP3, contributing to LUSC progression. Finally, tumor growth and metastasis were evaluated in vivo. STEAP3 expression was notably higher in LUSC, and its overexpression was linked to a poor prognosis. Moreover, STEAP3 overexpression activated the β-catenin pathway, thereby accelerating cell proliferation and metastasis. Conversely, STEAP3 knockdown had an anti-tumor effect in LUSC. Additionally, TFAP2C bound directly to the STEAP3 promoter and positively regulate its expression in LUSC. The anti-tumor effects of TFAP2C knockdown were partially reversed by STEAP3 overexpression. The study indicates that the TFAP2C/STEAP3 axis may be a therapeutic target for LUSC treatment. This enhances our understanding of lung carcinogenesis.
研究领域:
肿瘤学
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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
CELLULAR & MOLECULAR BIOLOGY LETTERS
·
2023
·Vol. 28
·期 1
·1-24
·
IF 8.3
Lei Ming,Lin Hui,Shi Deyao,Hong Pan,Song Hui,Herman Bomansaan,Liao Zhiwei,Yang Cao
Background Intervertebral disc degeneration (IVDD) is the major cause of low-back pain. Histone deacetylase 9 (HDAC9) was dramatically decreased in the degenerative nucleus pulposus (NP) samples of patients with intervertebral disc degeneration (IVDD) according to bioinformatics analysis of Gene Expression Omnibus (GEO) GSE56081 dataset. This study aims to investigate the role of HDAC9 in IVDD progression.Methods The contribution of HDAC9 to the progression of IVDD was assessed using HDAC9 knockout (HDAC9KO) mice and NP-targeted HDAC9-overexpressing mice by IVD injection of adenovirus-mediated HDAC9 under a Col2a1 promoter. Magnetic resonance imaging (MRI) and histological analysis were used to examine the degeneration of IVD. NP cells were isolated from mice to investigate the effects of HDAC9 on apoptosis and viability. mRNA-seq and coimmunoprecipitation/mass spectrometry (co-IP/MS) analysis were used to analyze the HDAC9-regulated factors in the primary cultured NP cells.Results HDAC9 was statistically decreased in the NP tissues in aged mice. HDAC9KO mice spontaneously developed age-related IVDD compared with wild-type (HDAC9WT) mice. In addition, overexpression of HDAC9 in NP cells alleviated IVDD symptoms in a surgically-induced IVDD mouse model. In an in vitro assay, knockdown of HDAC9 inhibited cell viability and promoted cell apoptosis of NP cells, and HDAC9 overexpression had the opposite effects in NP cells isolated from HDAC9KO mice. Results of mRNA-seq and co-IP/MS analysis revealed the possible proteins and signaling pathways regulated by HDAC9 in NP cells. RUNX family transcription factor 3 (RUNX3) was screened out for further study, and RUNX3 was found to be deacetylated and stabilized by HDAC9. Knockdown of RUNX3 restored the effects of HDAC9 silencing on NP cells by inhibiting apoptosis and increasing viability.Conclusion Our results suggest that HDAC9 plays an important role in the development and progression of IVDD. It might be required to protect NP cells against the loss of cell viability and apoptosis by inhibiting RUNX3 acetylation and expression during IVDD. Together, our findings suggest that HDAC9 may be a potential therapeutic target in IVDD.Graphical Abstract
研究领域:
骨科
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分子生物学
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治疗学
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生物信息学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）