Related Experiment Video
Updated: Dec 22, 2025
A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts
Published on: December 1, 2018
Non-negligible factors in studying the ApoM-S1P axis using EA.hy926 cells
Yuanping Shi1, Yun Liang2, Jun Zhang1
1Comprehensive Laboratory, the Third Affiliated Hospital of Soochow University, Changzhou 213003, China.
Background:
The apolipoprotein M (ApoM)-sphingosine-1-phosphate (S1P) axis was recently identified, and research into its function has received increasing attention. However, there are some factors which might influence the results of studies into the function of the ApoM-S1P axis using the EA.hy926 cells. This study investigated related factors, including coagulation factor VIII (FVIII), ApoM, S1P receptor subtypes (S1PRs), C-myc-tagged, and His-tagged proteins in EA.hy926 cells, as well as the effects of ApoM overexpression on S1PRs.
Methods:
The expression of FVIII, ApoM, S1PRs, C-myc, and His-tagged proteins in EA.hy926 cells was investigated through cellular immunofluorescence. EA.hy926 cells were infected with lentiviruses carrying (OE group) or lacking (NC group) the ApoM gene sequence. A stable cell line expressing ApoM was obtained, and the expression of ApoM mRNA was detected through single tube duplex fluorescence reverse transcription quantitative polymerase chain reaction (RT-qPCR). S1PRs expression was detected by RT-qPCR and Western blotting.
Results:
The results showed that EA.hy926 cells expressed FVIII, ApoM, C-myc-tagged, and His-tagged proteins. Moreover, they highly expressed S1PR1, slightly expressed S1PR3, weakly expressed S1PR2, and did not express S1PR4 and S1PR5. ApoM overexpression significantly increased S1PR1 mRNA and protein expression but did not affect the expression of S1PR3. EA.hy926 cells expressed FVIII, suggesting the cell line possesses endothelial cell characteristics and could be used for in vitro studies of the ApoM-S1P axis.
Conclusions:
EA.hy926 cell line is suitable for investigation of the ApoM-S1P axis in vitro. However, Since EA.hy926 cells expressed endogenous ApoM, C-myc and His tagged proteins, the exogenous recombinant ApoM should not be labeled with C-myc and His tags for distinguishing from endogenous ApoM. In addition, overexpression of ApoM should be considered to significantly increase the expression of S1PR1 when studying the APOM-S1P axis.
More Related Videos
11:35Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA
Published on: August 21, 2016
08:53Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings
Published on: July 15, 2019