肝癌细胞中[14C]色氨酸代谢示踪法

Krishna S. Tummala; Nabil Djouder

doi:10.21769/BioProtoc.1582

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
登录
/
注册
- 个人主页
- 编辑个人信息
- 修改密码
- 退出
CN
- EN - English
- CN - 中文

Peer-reviewed

[14C]-Tryptophan Metabolic Tracing in Liver Cancer Cells

肝癌细胞中[14C]色氨酸代谢示踪法

Krishna S. Tummala

Nabil Djouder email

发布: 2015年09月05日第5卷第17期 DOI: 10.21769/BioProtoc.1582 浏览次数: 10084

评审: Jia LiShannon RuppertYong Teng

PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Cancer Cell, featuring study using the protocol.

Dec 2014

Special Issue on Protocols for Organoid Research

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

Vangl 蛋白的泛素化和磷酸化分析

Di Feng [...] Bo Gao

2022年10月20日 3737 阅读

通过CRISPR-Cas9介导的HiBiT标签对高度同源蛋白水平进行异构体特异性半定量测定

Kristina Seiler [...] Mario P. Tschan

2023年07月20日 2777 阅读

Mag-Net 强阴离子交换技术用于分离卵巢癌腹水来源细胞外囊泡以开展蛋白质组学标志物发现

Tyler T. Cooper

2026年03月20日 506 阅读

Abstract

Nicotinamide adenine dinucleotide (NAD⁺) is a coenzyme for many NAD⁺-consuming proteins with diverse biological functions. Oscillations in NAD⁺ levels may influence several cellular signaling pathways. NAD⁺ synthesis via Preiss-Handler route (salvage reactions) has been extensively reported. However, the contribution of L-tryptophan/kynurenine catabolism in de novo NAD⁺ synthesis is poorly understood. Using L-[14C]-tryptophan tracing in four liver cancer cell lines and siRNA-mediated silencing of arylformamidase (AFMID), a key enzyme involved in L-tryptophan degradation, we demonstrate the contribution of L-tryptophan catabolism in de novo synthesis of NAD⁺ pools. NAD⁺ modulation is therefore important in maintaining cellular homeostasis and appropriate cellular functions according to nutrients availability.

Keywords: Kynurenine pathway (犬尿氨酸途径)

De novo NAD+ synthesis (从头合成NAD +)

[14C]-Tryptophan metabolic tracing ([14C] -色氨酸代谢示踪)

Arylformamidase (AFMID) (芳基甲酰胺酶（afmid）)

Liver cancer (肝癌)

Materials and Reagents

Liver cancer cell lines: Huh-7 (JCRB, catalog number: JCRB0403 ), HepG2 (ATCC, catalog number: HB-8065 ), SNU-398 (ATCC, catalog number: CRL-2233 ), and SNU-449 (ATCC, catalog number: CRL-2234 )
DMEM (Life technologies)
RPMI (Life technologies)
Amino acid free medium and without fetal bovine serum called Hanks’ Balanced Salt Solution (HBSS) (Sigma-Aldrich)
Fetal bovine serum (Cultek)
Penicilin and streptocmycin (Life Technologies, Gibco^®)
100x Non-essential amino acids’ solution (Life technologies)
RNAiMAX (Life Technologies, Invitrogen^TM, catalog number: 13778030 )
OptiMem (Life Technologies, catalog number: 11058021 )
Non silencing small interference RNA control (siCtl) and small interference RNA Arylformamidase (siAFMID) (GE Healthcare Dharmacon, catalog number: L-HUMAN-XX-0005 5 nmol )
NAD+-[carbonyl-14C] (Perkin Elmer, catalog number: NEC831010UC )
L-[benzene-ring-U-14C]-tryptophan (Moravek, catalog number: MC 2335 )
PEI-cellulose thin layer chromatography (TLC) (F plates of 20 x 20 cm) (Merk Millipore catalog number: 105725 )
Ammonium acetate (Sigma-Aldrich, catalog number: A1542 )
Ethanol and methanol (Sigma-Aldrich)
NAD⁺-[carbonyl-14C] (see Recipes)
L-[benzene-ring-U-14C]-tryptophan (see Recipes)
Cell density (around 60-80%) (see Recipes)

Equipment

Phosphor-image reader (Molecular Dynamics, model: Storm820 )
Phosphor-image screen (GE Healthcare Dharmacon, catalog number: 63-0034-79 )
Speedvac centrifuge (Thermo Fisher Scientific)
4 °C table top centrifuge (Eppendorf, model: 5424R )
Fume hood to handle radioactive materials
Cell scrapers (Corning Incorporated, catalog number: 3008 )
Ruler

Software

Fiji software (www.fiji.sc/Fiji)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Tummala, K. S. and Djouder, N. (2015). [14C]-Tryptophan Metabolic Tracing in Liver Cancer Cells. Bio-protocol 5(17): e1582. DOI: 10.21769/BioProtoc.1582.