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|Description||EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit|
|Overview||Unlike standard ChIP protocols that can be laborious and time consuming, the Magna ChIP kit protocol can reduce the amount of time required to perform a ChIP experiment from three days to one. Additionally, the smaller Magna ChIP reaction volume increases the relative concentration of the antibody enabling the ChIP reaction to be performed with reduced amounts of both antibody and sheared chromatin. Finally because this kit uses a blend of protein A and protein G beads, a wider range of antibody isotypes can be used than A or G alone. This allows a wider variety of antibodies to be used and avoids the need to purchase separate kits for protein A and protein G based immunoprecipitation. Because Magna ChIP kits use paramagnetic beads they are compatible with automated high throughput platforms, thus allowing a large number of ChIP reactions to be carried out simultaneously. Features & Benefits:
Chromatin Immunoprecipitation (ChIP) is an important technique allowing the analysis of in vivo interactions of proteins with genomic DNA. Any chromatin-associated or DNA binding protein can be analyzed with this technique, provided a good antibody to the protein exists. One can measure different proteins localized to a specific region of the genome, or the genome wide distribution of a specific protein. Another powerful application of this technique is to analyze changes in histone modifications that correlate with processes like transcription, mitosis or DNA repair.
|Materials Required but Not Delivered||Magna Grip™ Rack 8 well ( 20-400) (Now Available!) or similar magnetic rack.|
|Background Information||Chromatin Immunoprecipitation (ChIP) is a powerful technique for mapping the in vivo distribution of proteins associated with chromosomal DNA. These proteins can be histone subunits and post-translational modifications or other chromatin associated proteins such as transcription factors, chromatin regulators, etc. Additionally, ChIP can be used to identify regions of the genome associated with these proteins, or conversely, to identify proteins associated with a particular region of the genome. ChIP methodology often involves protein-DNA and protein-protein cross-linking, fragmentation of the cross-linked chromatin, and subsequent immunoprecipitation of chromatin with an antibody specific to a target protein. The DNA fragments isolated in complex with the target protein can be identified by a variety of methods including PCR, DNA microarray and DNA sequencing. Standard or quantitative PCR can be performed to verify whether a particular DNA sequence (the gene or region of the genome) is associated with the protein of interest. The combination of ChIP and promoter or genomic tiling microarrays (ChIP-chip) allows genome-wide identification of DNA-binding sites for chromatin-associated proteins with precise resolution. Alternatively, high-throughput sequencing of libraries constructed from immunoprecipitated chromosomal DNA (ChIP-Seq) is a powerful alternative to ChIP-chip in mapping the protein-DNA interactions across mammalian genomes.|
|Presentation||Two boxes containing all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions. Supplied buffers are sufficient to generate chromatin from up to five 15 cm plates of cultured cells, each plate providing up to 10 chromatin preparations (varies with cell and assay type).|
|Application||Single day chromatin immunoprecipitation (ChIP) kit containing all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions using magnetic A/G beads. Control primers included.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Upon receipt, store components at the temperatures indicated on the labels. Kit components are stable for 1 year from date of shipment when stored as directed.|
|Material Size||22 assays|
|Material Package||Kit capacity: 22 chromatin immunoprecipitation assays|
EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit SDS
|Reference overview||Pub Med ID|
|A functional CNVR_3425.1 damping lincRNA FENDRR increases lifetime risk of lung cancer and COPD in Chinese.|
Yang, L; Wu, D; Chen, J; Chen, J; Qiu, F; Li, Y; Liu, L; Cao, Y; Yang, B; Zhou, Y; Lu, J
Carcinogenesis 39 347-359 2018
Genomic imbalance referring to somatic variation in chromosome copies represents the most frequent event in tumorigenesis. Germline copy number variations (gCNVs) overlapping regions of genomic imbalance harbor similar structural characteristics and thus influence tumor susceptibility. We aimed to test effects of such gCNVs on the risk of lung cancer and chronic obstructive pulmonary disease (COPD). Genomic imbalance of lung cancer was determined by the array comparative genomic hybridization (aCGH), and common gCNVs at these imbalance regions were genotyped in lung cancer-based and COPD-based retrospective studies. Functional assays were conducted to assess function of promising CNVs. A total of 115 genomic imbalances were discovered occurring at a frequency of more than 25%. The CNVR_3425.1, overlapping the chr16q24.1 with genomic imbalance, was significantly associated with increased risks of lung cancer (OR = 1.76; 95% CI = 1.46-2.11) and COPD (OR = 1.98; 95% CI = 1.57-2.51). The increase copy of CNVR_3425.1 forms a new additional truncated FOXF1 adjacent non-coding developmental regulatory RNA (FENDRR) sequences comparing the gene promoter and perturbs the transcriptional factors (TFs) binding to the original FENDRR promoter and further downregulates FENDRR, a long intergenic non-coding RNA (lincRNA) that functions to inhibit lung cancer by affecting expressions of an abundant number of genes, including the tumor suppressor FOXF1. FENDRR can upregulate FOXF1 by competitively binding to miR-424. The TFs early growth response 1 (EGR1) and transcription factor AP-2 alpha (TFAP2A) were further found to involve the CNVR_3425.1-mediated FENDRR dysregulation. These findings suggested the CNVR_3425.1 to be a possibly predictive biomarker for the risk of lung cancer and COPD, and targeted molecular therapy pertaining to FENDRR upregulation may be a valuable pathway to fight two diseases.
|COX-2/PGE2 Axis Regulates HIF2α Activity to Promote Hepatocellular Carcinoma Hypoxic Response and Reduce the Sensitivity of Sorafenib Treatment.|
Dong, XF; Liu, TQ; Zhi, XT; Zou, J; Zhong, JT; Li, T; Mo, XL; Zhou, W; Guo, WW; Liu, X; Chen, YY; Li, MY; Zhong, XG; Han, YM; Wang, ZH; Dong, ZR
Clin Cancer Res 0 2018
Purpose: Hypoxia-inducible factor-2α (HIF2α) is regarded as a preferential target for individualized hepatocellular carcinoma (HCC) treatment and sorafenib resistance. Our study aimed to identify the regulatory mechanisms of HIF2α activity under hypoxic conditions. We sought to determine whether the COX-2/PGE2 axis is involved in the regulatory mechanisms of HIF2α activity and of sorafenib resistance in hypoxic HCC cells.Experimental design: The cell viability, migration, and invasion abilities were measured to analyze the effects of HIF2α on hypoxic HCC cells. Both in vitro and in vivo HCC models were used to determine whether the COX-2/PGE2 axis is a driver of HIF2α level and activity, which then reduces the sensitivity of sorafenib treatment in hypoxic HCC cells.Results: Under hypoxic conditions, the COX-2/PGE2 axis effectively stabilized HIF2α and increased its level and activity via decreasing von Hippel-Lindau protein (p-VHL) level, and also enhanced HIF2α activity by promoting HIF2α nuclear translocation via MAPK pathway. The activation of HIF2α then led to the enhanced activation of VEGF, cyclin D1, and TGFα/EGFR pathway to mediate HCC development and reduce the sensitivity of sorafenib. More importantly, COX-2-specific inhibitors synergistically enhanced the antitumor activity of sorafenib treatment.Conclusions: Our data obtained demonstrate that the COX/PGE2 axis acts as a regulator of HIF2α expression and activity to promote HCC development and reduce sorafenib sensitivity by constitutively activating the TGFα/EGFR pathway. This study highlights the potential of COX-2-specific inhibitors for HCC treatment and particularly for enhancing the response to sorafenib treatment. Clin Cancer Res; 1-13. ©2018 AACR.
|Aberrant methylation-mediated silencing of lncRNA CTC-276P9.1 is associated with malignant progression of esophageal squamous cell carcinoma.|
Guo, W; Liu, S; Dong, Z; Guo, Y; Ding, C; Shen, S; Liang, J; Shan, B
Clin Exp Metastasis 35 53-68 2018
Downregulation and aberrant hypermethylation of long non-coding RNA CTC-276P9.1 have been detected in limited tumors. However, the distribution of methylated CpG sites and biological role of CTC-276P9.1 in esophageal squamous cell carcinoma (ESCC) progression and prognosis have not been fully clarified. The present study was to investigate the expression status and the distribution of methylated CpG sites within the three CpG islands of CTC-276P9.1, further to clarify its functional role and prognostic value in ESCC development and prognosis. Significant downregulation of CTC-276P9.1 was detected in esophageal cancer cells and ESCC tissues, and the expression of CTC-276P9.1 in ESCC tissues was associated with TNM stage, pathological differentiation, lymph node metastasis, and distant metastasis or recurrence. The expression level of CTC-276P9.1 in esophageal cancer cells was significantly reversed by treatment with 5-Aza-dC and TSA. The aberrant hypermethylation of the regions around the transcription start site was more tumor specific and associated with the expression levels of CTC-276P9.1. Moreover, histone modification may also participate in the regulation of CTC-276P9.1. Furthermore, over-expression of CTC-276P9.1 inhibited esophageal cancer cells proliferation and invasion in vitro, decreased the expression of proliferative markers and inhibited esophageal cancer cells invasion probably by regulating EMT. In addition, the dysregulation and hypermethylation of the regions around the transcription start site of CTC-276P9.1 were associated with poorer ESCC patients' survival. These findings suggest that CTC-276P9.1 may act as a tumor suppressor and may be employed as a new prognostic factor and therapeutic target for ESCC.
|BRD4 Inhibition Is Synthetic Lethal with PARP Inhibitors through the Induction of Homologous Recombination Deficiency.|
Sun, C; Yin, J; Fang, Y; Chen, J; Jeong, KJ; Chen, X; Vellano, CP; Ju, Z; Zhao, W; Zhang, D; Lu, Y; Meric-Bernstam, F; Yap, TA; Hattersley, M; O'Connor, MJ; Chen, H; Fawell, S; Lin, SY; Peng, G; Mills, GB
Cancer Cell 33 401-416.e8 2018
Poly(ADP-ribose) polymerase inhibitors (PARPi) are selectively active in cells with homologous recombination (HR) deficiency (HRD) caused by mutations in BRCA1, BRCA2, and other pathway members. We sought small molecules that induce HRD in HR-competent cells to induce synthetic lethality with PARPi and extend the utility of PARPi. We demonstrated that inhibition of bromodomain containing 4 (BRD4) induced HRD and sensitized cells across multiple tumor lineages to PARPi regardless of BRCA1/2, TP53, RAS, or BRAF mutation status through depletion of the DNA double-stand break resection protein CtIP (C-terminal binding protein interacting protein). Importantly, BRD4 inhibitor (BRD4i) treatment reversed multiple mechanisms of resistance to PARPi. Furthermore, PARPi and BRD4i are synergistic in multiple in vivo models.
|Aberrant methylation-mediated downregulation of long noncoding RNA C5orf66-AS1 promotes the development of gastric cardia adenocarcinoma.|
Guo, W; Lv, P; Liu, S; Xu, F; Guo, Y; Shen, S; Liang, J; Kuang, G; Dong, Z
Mol Carcinog 57 854-865 2018
As a long non-coding RNA, C5orf66-AS1 is located at 5q31.1. Downregulation and aberrant hypermethylation of C5orf66-AS1 have been detected in a limited several tumors. However, the biological role and distribution of methylated CpG sites of C5orf66-AS1 in gastric cardia adenocarcinoma (GCA) development and prognosis are poorly clarified. The present study was to investigate the expression status and function of C5orf66-AS1 in GCA, and to detect the distribution of methylated CpG sites within the three CpG islands of the promoter and gene body of C5orf66-AS1, further to clarify its prognostic value in GCA patients. C5orf66-AS1 was significantly downregulated in GCA tissues and cell lines, and the expression level was associated with TNM stage, pathological differentiation, lymph node metastasis, and distant metastasis or recurrence. The expression level of C5orf66-AS1 was significantly increased in cancer cells after treated with 5-Aza-dC. Further methylation analysis demonstrated that the aberrant hypermethylation of the regions around the transcription start site of C5orf66-AS1 was more tumor specific and was associated with its expression. Moreover, Sp1 may upregulate C5orf66-AS1 expression and CpG sites hypermethylation within the binding sites may abrogate Sp1 binding. In addition, C5orf66-AS1 inhibited gastric cancer cell proliferation and invasion, and the dysregulation and hypermethylation of the regions around the transcription start site of C5orf66-AS1 were associated with poorer GCA patients' survival. These findings suggest that aberrant hypermethylation-mediated downregulation of C5orf66-AS1 may play important roles in GCA tumorigenesis and C5orf66-AS1 may serve as a potential prognostic marker in predicting GCA patients' survival.
|Long noncoding RNA BLACAT2 promotes bladder cancer-associated lymphangiogenesis and lymphatic metastasis.|
He, W; Zhong, G; Jiang, N; Wang, B; Fan, X; Chen, C; Chen, X; Huang, J; Lin, T
J Clin Invest 128 861-875 2018
The prognosis for bladder cancer patients with lymph node (LN) metastasis is dismal and only minimally improved by current treatment modalities. Elucidation of the molecular mechanisms that underlie LN metastasis may provide clinical therapeutic strategies for LN-metastatic bladder cancer. Here, we report that a long noncoding RNA LINC00958, which we have termed bladder cancer-associated transcript 2 (BLACAT2), was markedly upregulated in LN-metastatic bladder cancer and correlated with LN metastasis. Overexpression of BLACAT2 promoted bladder cancer-associated lymphangiogenesis and lymphatic metastasis in both cultured bladder cancer cell lines and mouse models. Furthermore, we demonstrate that BLACAT2 epigenetically upregulated VEGF-C expression by directly associating with WDR5, a core subunit of human H3K4 methyltransferase complexes. Importantly, administration of an anti-VEGF-C antibody inhibited LN metastasis in BLACAT2-overexpressing bladder cancer. Taken together, these findings uncover a molecular mechanism in the lymphatic metastasis of bladder cancer and indicate that BLACAT2 may represent a target for clinical intervention in LN-metastatic bladder cancer.
|Ehrlichia chaffeensis TRP120 Effector Targets and Recruits Host Polycomb Group Proteins for Degradation To Promote Intracellular Infection.|
Mitra, S; Dunphy, PS; Das, S; Zhu, B; Luo, T; McBride, JW
Infect Immun 86 2018
Ehrlichia chaffeensis has a group of well-characterized type I secreted tandem repeat protein (TRP) effectors that have moonlighting capabilities. TRPs modulate various cellular processes, reprogram host gene transcription as nucleomodulins, function as ubiquitin ligases, and directly activate conserved host cell signaling pathways to promote E. chaffeensis infection. One TRP-interacting host target is polycomb group ring finger protein 5 (PCGF5), a member of the polycomb group (PcG) protein family and a component of the polycomb repressive complex 1 (PRC1). The current study demonstrates that during early infection, PCGF5 strongly colocalizes with TRP120 in the nucleus and later dramatically redistributes to the ehrlichial vacuole along with other PCGF isoforms. Ectopic expression and immunoprecipitation of TRP120 confirmed the interaction of TRP120 with multiple different PCGF isoforms. At 48 h postinfection, a dramatic redistribution of PCGF isoforms from the nucleus to the ehrlichial vacuole was observed, which also temporally coincided with proteasomal degradation of PCGF isoforms and TRP120 expression on the vacuole. A decrease in PRC1-mediated repressive chromatin mark and an altered transcriptional activity in PRC1-associated Hox genes primarily from HOXB and HOXC clusters were observed along with the degradation of PCGF isoforms, suggesting disruption of the PRC1 in E. chaffeensis-infected cells. Notably, small interfering RNA (siRNA)-mediated knockdown of PCGF isoforms resulted in significantly increased E. chaffeensis infection. This study demonstrates a novel strategy in which E. chaffeensis manipulates PRC complexes through interactions between TRP120 and PCGF isoforms to promote infection.
|The effects of wild bitter gourd fruit extracts on ICAM-1 expression in pulmonary epithelial cells of C57BL/6J mice and microRNA-221/222 knockout mice: Involvement of the miR-221/-222/PI3K/AKT/NF-κB pathway.|
Sung, HC; Liu, CW; Hsiao, CY; Lin, SR; Yu, IS; Lin, SW; Chiang, MH; Liang, CJ; Pu, CM; Chen, YC; Lin, MS; Chen, YL
Phytomedicine 42 90-99 2018
The extracts from wild bitter gourd fruit (WBGE) were reported to possess numerous pharmacological activities. However, the anti-inflammatory effects of WBGE on human lung epithelial cells and the underlying mechanisms have not been determined.To evaluate the molecular basis of the effects of WBGE on intercellular adhesion molecule-1 (ICAM-1) expression in alveolar epithelial (A549) cells, C57BL/6 wild-type (WT) mice and microRNA (miR)-221/-222 knockout (KO) mice with or without tumor necrosis factor (TNF-α; 3 ng/ml) treatment.WT mice and miR-221/-222 KO mice were fed a control diet and divided into four groups (C: control mice; T: treated with TNF-α alone; WBGE/T: pretreated with WBGE and then stimulated with TNF-α; WBGE: treated with WBGE alone). The effects of WBGE on ICAM-1 expression and the related signals in A549 cells and mice with or without TNF-α treatment were examined by Western blot and immunofluorescent staining.WBGE significantly decreased the TNF-α-induced ICAM-1 expression in A549 cells through the inhibition of phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT)/ nuclear factor- kappa B (NF-κB)/ inhibitor of NF-κB (IκB) phosphorylation and decreased leukocyte adhesion. In addition, WBGE reduced endogenous ICAM-1 expression and upregulated miR-221/-222 expression. The overexpression of miR-222 decreased PI3K/AKT/NF-κB/IκB and ICAM-1 expression, which resulted in reducing monocyte adhesion. Moreover, WBGE reduced ICAM-1 expression in lung tissues of WT mice with or without TNF-α treatment and upregulated miR-221/222. WBGE did not affect the miR-221/-222 level and had little effect on ICAM-1 expression in miR-221/-222 KO mice.These results suggest that WBGE reduced ICAM-1 expression both under in vitro and in vivo conditions. The protective effects were mediated partly through the miR-221/-222/PI3K/AKT/NF-κB pathway.
|LncRNA FIRRE/NF-kB feedback loop contributes to OGD/R injury of cerebral microglial cells.|
Zang, Y; Zhou, X; Wang, Q; Li, X; Huang, H
Biochem Biophys Res Commun 501 131-138 2018
Stroke is one of the leading causes for serious long-term neurological disability. LncRNAs have been investigated to be dysregulated in ischemic stroke. However, the underlying mechanisms of some specific lncRNAs have not been clearly clarified. To determine lncRNA-mediated regulatory mechanism in ischemic stroke, we constructed OGD/R injury model of cerebral microglial cells. Microarray analysis was carried out and analyzed that lncRNA functional intergenic repeating RNA element (FIRRE) was associated with OGD/R injury. Based on the molecular biotechnology, we demonstrated that FIRRE could activate NF-kB signal pathway. Meanwhile, the activated NF-kB promoted FIRRE expression in OGD/R-treated cerebral microglial cells. Therefore, FIRRE and NF-kB formed a positive feedback loop to promote the transcription of NLRP3 inflammasome, thus contributed to the OGD/R injury of cerebral microglial cells. All findings in this study may help to explore novel and specific therapeutic target for ischemic stroke.
|Glycine N-methyltransferase inhibits aristolochic acid nephropathy by increasing CYP3A44 and decreasing NQO1 expression in female mouse hepatocytes.|
Chang, MM; Lin, CN; Fang, CC; Chen, M; Liang, PI; Li, WM; Yeh, BW; Cheng, HC; Huang, BM; Wu, WJ; Chen, YA
Sci Rep 8 6960 2018
Plants containing aristolochic acids (AA) are nephrotoxins. Glycine N-methyltransferase (GNMT) acts to bind environmental toxins such as benzo(a)pyrene and aflatoxin B1, translocate into nucleus, and alter hepatic metabolism. This study aims to determine the role of GNMT in AA-induced nephropathy. We established an AA nephropathy mouse model and found that AA type I (AAI)-induced nephropathy at a lower concentration in male than in female mice, implying sex differences in AAI resistance. Microarray analysis and AAI-treated mouse models showed that GNMT moderately reduced AAI-induced nephropathy by lowering the upregulated level of NQO1 in male, but significantly improved the nephropathy additionally by increasing Cyp3A44/3A41 in female. The protective effects of GNMT were absent in female GNMT knockout mice, in which re-expression of hepatic GNMT significantly decreased AAI-induced nephropathy. Mechanism-wise, AAI enhanced GNMT nuclear translocation, resulting in GNMT interaction with the promoter region of the genes encoding Nrf2 and CAR/PXR, the transcription factors for NQO1 and CYP3A44/3A41, respectively. Unlike the preference for Nrf2/NQO1 transcriptions at lower levels of GNMT, overexpression of GNMT preferred CAR/PXR/CYP3A44/3A41 transcriptions and alleviated kidney injury upon AAI treatment. In summary, hepatic GNMT protected mice from AAI nephropathy by enhancing CAR/PXR/CYP3A44/3A41 transcriptions and reducing Nrf2/NQO1 transcriptions.
|White Paper - The Message in the Marks: Deciphering Cancer Epigenetics (EMD)|
Instructions for Use
|EZ-Magna ChIP A/G One-Day Chromatin Immunoprecipitation Kits|