RNAScope as a platform to detect IDO1 expression in tumor tissue sections.

2018 ◽  
Vol 36 (5_suppl) ◽  
pp. 116-116 ◽  
Author(s):  
Michael Pratta
Keyword(s):  
Western Blot ◽  
Hela Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Tumor Tissue ◽  
Activity Level ◽  
Multiple Tumor ◽  
Tissue Sections ◽  
Ido1 Expression ◽  
Tumor Types

116 RNAScope is a sensitive, specific platform to detect IDO1 expression in tumor tissue sections. M Pratta, M Rupar, P Waeltz, T Burn, G Hollis, M Covington, M Smith, and R Newton. Incyte Corp. Wilm. DE. Background: Indoleamine 2,3-Dioxygenase 1 (IDO1) catalyzes the primary and rate-limiting step in tryptophan catabolism to generate N-formyl-kynurenine (Kyn). Through a combination of local depletion of tryptophan and an increase in Kyn concentrations, IDO-1 activity can result in the suppression of antitumor immune responses. Because IDO-1 inhibitors are now in the clinic for treatment of multiple tumor types, immunohistological approaches are employed to demonstrate IDO1 expression in tumor biopsies. However, using a commercially available antibody to detect IDO1 by immunohistochemistry (IHC), the level of sensitivity was inadequate. Methods: In order to improve the sensitivity of IDO1 detection, we evaluated in situ hybridization (ISH) using RNAScope technology and digital quantitation by HALO analysis in collaboration with Advanced Cell Diagnostics (ACD). The technology was cross-validated using IDO1 qRT-PCR, Western blot, and activity analysis and compared with standard IHC. We initially evaluated IDO1 expression in HeLa cells stimulated with various concentrations of IFNγ, and then extended the observations using tissue sections from multiple tumor types. Results: In the HeLa cell model, IFNγ induced a time- and concentration-dependent increase of IDO1 at the mRNA, protein, and activity level. Although IDO1 was successfully detected in the HeLa cell samples by IHC, comparison of the platforms indicated IFNγ EC50 values were in strong agreement between RNAScope (193.8 pg/ml) and Western blot analysis (170.8 pg/ml), but was much higher by IHC analysis (2206 pg/ml). A strong positive correlation (*p < 0.0001) between RNAScope and Western blot analysis was observed, suggesting a highly coordinated induction of IDO1 by IFNγ at both the mRNA and protein levels. FFPE tumor tissue from melanoma, HNSCC, bladder, renal, ovarian, and lung cancers visualized by RNAScope all show varying levels of IDO1 expression. Conclusions: These data support the use of RNAScope for the analysis of IDO1 expression in clinical trials.

scholarly journals Western blot analysis of HeLa cell ferritin.

1991 ◽  
Vol 35 (6) ◽  
pp. 441-444
Author(s):  
Keiko Yamaguchi
Keyword(s):  
Western Blot ◽  
Hela Cell ◽  
Blot Analysis ◽  
Western Blot Analysis

scholarly journals Extraction and Analysis of Diagnostically Useful Proteins from Formalin-fixed, Paraffin-embedded Tissue Sections

1998 ◽  
Vol 46 (3) ◽  
pp. 397-403 ◽  
Author(s):  
Kimimasa Ikeda ◽  
Takushi Monden ◽  
Toshiyuki Kanoh ◽  
Masaki Tsujie ◽  
Hikaru Izawa ◽  
...  
Keyword(s):  
Western Blot ◽  
Cyclin D1 ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Protein Extraction ◽  
Simple Method ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-μm-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as β-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.

Abstract P071: Transglutaminases Are Active In Perivascular Adipose Tissue

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Alexis Orr ◽  
Janice Thompson ◽  
Janae Lyttle ◽  
Stephanie W Watts
Keyword(s):  
Adipose Tissue ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Coagulation Factor ◽  
Sprague Dawley ◽  
Perivascular Adipose Tissue ◽  
Tissue Sections ◽  
Mesenteric Resistance Artery ◽  
Immunohistochemical Analyses

Transglutaminases (TGs) are crosslinking enzymes best known for their vascular remodeling in hypertension. They require calcium to form an isopeptide bond, connecting a glutamine to a protein bound lysine residue or a free amine donor such as norepinephrine (NE) or serotonin (5-HT). We discovered that perivascular adipose tissue (PVAT) contains significant amounts of these amines, making PVAT an ideal model in which to test interactions of amines and TGs. We hypothesized that TG2 and FXIII are active in PVAT. Sprague-Dawley rat aortic, superior mesenteric (SMA), and mesenteric resistance artery (MR) PVAT express TG2 and blood coagulation factor XIII (FXIII) mRNA (Figure 1A). Consistent with this, immunohistochemical analyses support that PVATs all express TG2 and FXIII protein. The activity of TG2 and FXIII was investigated in tissue sections using substrate peptides that label active TGs and a catalyzing calcium solution, visualized with TRITC fluorescence (Figure 1B,C). Both TG2 and FXIII are active in rat aortic PVAT, SMAPVAT, and MRPVAT. Western blot analysis determined that the known TG inhibitor cystamine reduced incorporation of experimentally added amine donor 5-(biotinamido)pentylamine (BAP) into MRPVAT by 6.14% of total normalized signal (p<0.0001, N=7). Further Western blot analysis proved that experimentally added 5-HT competitively inhibits incorporation of experimentally added BAP into MRPVAT adipocytes, reducing total normalized signal by 10.75% (p=0.001, N=4). Further studies to determine what proteins TGs are amidating will give insight into how these enzymes contribute to the development of hypertension.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

2020 ◽  
Vol 20 (9) ◽  
pp. 1147-1156
Author(s):  
Hanrui Li ◽  
GeTao Du ◽  
Lu Yang ◽  
Liaojun Pang ◽  
Yonghua Zhan
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Tumor Size ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Bioluminescence Imaging ◽  
Antitumor Effects ◽  
In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

scholarly journals Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2794 ◽  
Author(s):  
Cao ◽  
Chen ◽  
Ren ◽  
Zhang ◽  
Tan ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Responses ◽  
Raw264.7 Macrophages ◽  
Tnf Α ◽  
Autophagy Inhibition ◽  
Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

scholarly journals Protocol for determining protein cysteine thiol redox status using western blot analysis

2021 ◽  
Vol 2 (2) ◽  
pp. 100566
Author(s):  
Bikram Datt Pant ◽  
Sunhee Oh ◽  
Kirankumar S. Mysore
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Redox Status ◽  
Thiol Redox
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