Non-negligible factors in studying the ApoM-S1P axis using EA.hy926 cells

Abstract

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.

Genetically Programmable Vesicles for Enhancing CAR‐T Therapy against Solid Tumors

“…To obtain cellular vesicles expressing murine anti‐PD‐L1 antibody (C5H9v2), we first designed the sequence expressing murine aPD‐L1 single chain variable fragment (scFv) on the cell membrane ( Figure a); the accompanying MYC protein is often utilized as a tag in Western blotting. [ 34 ] Then, we subcloned the sequence encoding aPD‐L1 scFv into a lentiviral vector containing a green fluorescent protein (GFP) and co‐transformed into HEK 293T with helper plasmids. HEK 293T is often regarded as an engineering cell line, which can easily express exogenous recombinant proteins and retrovirus after transfection with plasmids for lentivirus production.…”