Establishment of cell-based screening system for compound regulating the stability of retinoic acid receptors
10.3969/j.issn.1674-8115.2017.04.002
- VernacularTitle:调节维甲酸受体稳定性的化合物细胞筛选体系的建立
- Author:
Bo JING
;
Penghui CHEN
;
Xiang GAO
;
Yuanyuan XU
;
Yunzhao WU
;
Yun SUN
;
Yingli WU
- Keywords:
RARα;
drug screening;
sodium valproate;
all-trans retinoic acid;
NB4 cell
- From:
Journal of Shanghai Jiaotong University(Medical Science)
2017;37(4):432-437
- CountryChina
- Language:Chinese
-
Abstract:
Objective·To establish a cell-based screening system for identification of compounds with activity in regulating retinoic acid receptor (RARα) stability. Methods·The modified pMSCV plasmid constructs, named as RARα-EGFP-IRES-DsRed, consists of enhanced green fluorescent protein (EGFP) fusing to RARα and red fluorescent protein (DsRed) as internal references incorporating the internal ribosome entry site (IRES) as interval sequence. The RARα-EGFP-IRES-DsRed plasmid was stably transfected into NB4 cells which were named as NB4-pMGIR-RARα. Fluorescence signals of EGFP and DsRed indirectly reflecting the expression of RARα, were detected by flow cytometry in cells that were treated with all-trans retinoic acid, sodium valproate, cytarabine, lenalidomide, etoposide, montelukast and gambogic acid, respectively. Effects of these compounds on the expression of RARα protein were further examined by Western blotting. Results·A double fluorescence reporter system for screening compounds that can increase the stability of RARα protein was successfully established, and sodium valproate was identified as a potent compound to promote the stability of RARα. Conclusion·The double fluorescence reporter system can be used to screen compounds regulating the stability of RARα protein, which can be further used to identify compounds regulating the stability of other proteins.
