Ex neg m02 b

The empty control mammalian expression plasmid pReceiver-M02 (#EX-NEG-M02-B), and plasmid vectors for AUF1 (#EX-Z0257-M02-B), ELAVL1 (#EX-Q0365-M02-B), and KHSRP (#EX-Z9878-M02-B) were purchased from Genecopoeia. These vectors were co-transfected with non-risk (rs3045215 non-deletion allele) and risk (rs3045215 deletion allele) luciferase reporters in HEK293 cells using the Lipofectamine 3000 kit (Invitrogen). The luciferase reporters were described previously (12 (link)). HEK293 cells were plated at 1 × 10⁶/ml, 1 ml per well, in 12-well-plates and allowed to adhere overnight. The next day, cells were transfected with 1 μg of luciferase reporter (either non-deletion or deleted) in triplicate together with the empty pEZ vector control or with 10, 50, or 100 ng of each RBP vector (either AUF1, KHSRP, or ELAVL1). Each experiment was repeated 5–7 times. Overexpression of each vector in HEK293 cells was confirmed by immunoblot. HEK293 cells were grown in 6-well-plates and maintained in DMEM (low glucose, Gibco) medium with 10% FBS and Pen-Strep.

To induce stromal cells to undergo decidualization, the harvested stromal cells were cultured for 30 min, the medium was changed to remove unattached cells. And the cell culture was continued by adding fresh medium supplemented with P4 (1 μM) and E2 (10 nM) dissolved in ethanol for different time points. Transfection of Myc overexpression plasmid in Mettl3 cKO uterine stromal cells was performed according to Lipo3000 protocol (L3000015, Life Technologies). For the six-well culture plate, 2 μg Myc overexpression plasmid (EX-Mm30812-M02, GeneCopoeia) or 2 μg control plasmid (EX-NEG-M02-B, GeneCopoeia) was used for the transfection. The cells were cultured with P4 (1 μM) and E2 (10 nM) for 2 days, and the mRNA levels of Myc and of decidualization-related genes were analyzed by RT-qPCR.