A rapid detection method for activated RhoA proteins based on high-content image anylysis
10.3867/j.issn.1000-3002.2024.11.005
- VernacularTitle:一种基于高内涵成像分析技术的快速检测RhoA蛋白激活的方法
- Author:
Yanan ZHOU
1
;
Ying QU
;
Shaowen WANG
;
Yi SUN
;
Ruibin SU
Author Information
1. 南京中医药大学,江苏 南京 210023;军事医学研究院国家安全特需药品全国重点实验室,神经精神药理学北京市重点实验室,北京 100850
- Keywords:
RhoA;
protein detection;
high-content imaging analysis;
fluorescent staining;
high-throughput
- From:
Chinese Journal of Pharmacology and Toxicology
2024;38(11):839-845
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE To develop a rapid method for detection of activated RhoA protein using the high content imaging system(HCIS).METHODS Hek293 or CHO cells were seeded in 96-well plates and subjected to starvation treatment after attachment.Hek293 cells were incubated with nocodazole,a RhoA agonist,at concentrations of 0(vehicle control),10-11,10-10,10-9,10-8,10-7,10-6,10-5 and 10-4 mol·L-1 for 3,10 and 30 min respectively.CHO cells were incubated with nocodazole,lyso-phosphatidic acid(LPA)and calpain at the same concentrations for 3,10 and 30 min respectively.Imme-diately after incubation,the cells were fixed with 3.7%formaldehyde solution and stained using Hoechst and rhodamin phallodin at room temperature and protected from light.Images were captured using HCIS and analyzed statistically.Changes in the mean fluorescence intensity(MFI)were used to assess the activation of RhoA protein by the drugs.RESULTS Compared with the vehicle control group,the MFI of Hek293 cells treated with nocodazole for 3 min significantly increased at concentra-tions ranging from 10-10 to 10-6 mol·L-1(P<0.01).When the treatment duration was extended to 30 min,MFI elevations were observed at concentrations between 10-10 and 10-4 mol·L-1(P<0.01),indicating the activation of RhoA protein.In CHO cells,compared with the vehicle control group,MFI was increased after 10-10-10-6 mol·L-1 nocodazole treatment of 10 min and 30 min(P<0.05,P<0.01).Similarly,MFI was also increased under various conditions of LPA and calpeptin treatment.LPA 10-11-10-4 mol·L-1 treatment of 3 min and 10-11,10-8-10-4 mol·L-1 treatment of 10 min and 10-11-10-9,10-7,10-6,10-4 mol·L-1 treatment of 30 min all resulted in an elevated MFI(P<0.05,P<0.01).Calpeptin 10-11-10-6,10-4 mol·L-1 treatment of 10 min and 10-11 and 10-4 mol·L-1 treatment of 30 min also resulted in an elevated MFI(P<0.05,P<0.01).These results indicated that RhoA protein was effectively activated.CONCLUSION A method for rapid detection of RhoA protein activation has been established,which is capable high-throughput,rapid and easy detection of activated RhoA protein.
