Lipo3000

Cells were transfected with siRNAs-Tlr2, siRNA-Beclin1 or with control siRNA using Lipofectamine® 3000 (Lipo3000, Thermo Fisher Scientific, Inc., MA, USA) at 37 °C in a humidified incubator with 5% CO₂. The siRNAs were designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Another group of cells was transfected with a GFP-labeled nonspecific siRNA that served as the negative control (NC). The sequences of the siRNAs used in the present study were as follows: siRNA-Tlr2 sense: 5′-CAG AUC UAC AGA GCU AUG ATT-3′, anti-sense: 5′-UCA UAG CUC UGU AGA UCU GTT-3′; siRNA-Beclin1 sense: 5′-GUG GAA UGG AAU GAG AUU ATT-3′, anti-sense: 5′-UAA UCU CAU UCC AUU CCA CTT-3′; NC-siRNA sense: 5′-UUC UCC GAA CGU GUC ACG UTT-3′, anti-sense: 5′-ACG UGA CAC GUU CG GAG AAT T -3′. When NPCs seeded into 6-well plates reached 80% confluence, transfection was performed by mixing 5 µL siRNA with 5 µL Lipo3000 in a final volume of 2000 µL DMED/F12 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 15% serum without antibiotics, according to the manufacturer’s protocol. After 16 h of transfection, the cells were infected with P. acnes for 8 h. Finally, the mRNA and protein were extracted from the cells. Transfections were performed in triplicate, and the experiment was repeated at least three times.

GenePharma discovered and produced siRNAs that target SNX5 (m-Pack1999, RIBOBIO, Guangzhou, China). The sequence of siRNA was as follows: siRNA#1, 5′GGATGACTTCTTTGAGCAA 3 ′; siRNA#2, 5 ′ GCACAAAGGCCCTAATTGA3 ′; siRNA#3, 5′ GGAAGAGAGTGGCAGCATT3′. Lipofectamine 3000 (Lipi3000, L3000015, Invitrogen, CA, USA) was used to transfect the siRNA to the cells according to the manufacturer's protocol. Lipo3000 and siRNA were diluted for in OptiMEM (31985070, Gibco, CA, USA) and mixed gently; then, the mixture was added to the cell cultures.

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.