NFAT-Luc Signaling Assay in rPAECs

To assay the signal transduction of Ca²⁺/calcineurin/NFAT signaling, the commercial recombinant NFAT-Luc plasmids (Yesen, Shanghai, China) were purchased and transfected into rPAECs by using Lipofectamine 2000 (Invitrogen). Then, the transfected rPAECs were divided into six groups for indicated treatment: Nx, Hx, Hx + lv-shNC, Hx + lv-shMETTL3, Hx + HF, and Hx + lv-shMETTL3+HF. After 24 h, the cells were harvested to determine the luciferase activity using a dual luciferase reporter assay system (Promega, Madison, WI, USA).

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Kong C., Zhang F., Hu R, & Wang L. (2023). METTL3 Promotes Endothelium-Mesenchymal Transition of Pulmonary Artery Endothelial Cells by Regulating TRPC6/Calcineurin/NFAT Signaling Pathways. Evidence-based Complementary and Alternative Medicine : eCAM, 2023, 8269356.

Publication 2023

Lipofectamine 2000 dual luciferase reporter assay system

Assay Calcineurin Cells Lipofectamine 2000 Luciferase Plasmids Promega

Corresponding Organization : Hunan Provincial People's Hospital

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Variable analysis

independent variables

Treatment condition: Nx, Hx, Hx + lv-shNC, Hx + lv-shMETTL3, Hx + HF, and Hx + lv-shMETTL3+HF

dependent variables

Luciferase activity

control variables

Transfection of rPAECs with NFAT-Luc plasmids
Incubation time of 24 hours

controls

Positive control: Not specified
Negative control: Nx (normoxia) condition

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