Allstars neg sirna af 488

Culture media and supplements were purchased from Gibco. Chemical reagents and chemical inhibitors were purchased from Sigma and Calbiochem, respectively. TLR ligands, including poly I:C (tlr-pic), cl097 (tlrl-c97-5), ODN2395 (tlrl-2395), Pam3CSK4 (tlrl-pms), and LPS (tlrl-3pelps) were purchased from InvivoGen. Murine or human macrophage colony-stimulating factor (M-CSF) and ELISA Kits (TNF, IL-6, IFN-λ2/3, CCL2, CCL3, CCL5, and IP-10) was obtained from R&D Systems. Anti-phospho-TBK1 (#5483), anti-TBK1 (#3504), anti-phospho-IRF3 (#4947), anti-IRF3 (#4302), anti-phospho-IκBα (#9246), and anti-IκBα (#4814) Abs were purchased from Cell Signaling Technology. TLR3 antibody (#GTX20260) and TLR7 antibody (#NBP2-24906) were purchased from Genetex and Novus Biologicals. TLR3/dsRNA complex inhibitor (#614310) was purchased from Calbiochem Merck Millipore. The transfection reagents used were as follows: ON-TARGETplus Non-targeting Control Pool (Dharmacon, D-001810-10-50), ON-TARGETplus Human TLR3 siRNA-SMARTpool (Dharmacon, L-007745-00-0020), HiPerFect Transfection Reagent (Qiagen, 301707), human CLEC18 siRNA#1 (Qiagen, SI03231088), human CLEC18 siRNA#2 (Qiagen, SI04149502), AllStars Negative Control siRNA (Qiagen, 1027280), and AllStars Neg.siRNA AF 488 (Qiagen, 1027289).

For let-7c-5p upregulation, a let-7c miRNA mimic (miScript miRNA Mimics, Qiagen) was transfected in 2 N-HFF lines. Cells were plated in a concentration of 70,000/well on 24 well plates (BD Falcon) and after 24 hours were transfected with a miRNA mimic using the INTERFERin transfection reagent (Polyplus-transfection). A fluorescent siRNA (AllStars Neg. siRNA AF 488, Qiagen) has been used to monitor the efficiency of the chosen transfecting agent. Forty-eight hours after the transfection cells were harvested and ANT1 and DICER expression were evaluated, cells treated with the INTERFERin transfection agent only were used as mock control for all experiments performed after transfection.

Cultured HTR-8/SV-neo and JEG-3 (2×10⁵ cells/well in 6-well plates and 5×10⁴ cells/well in 24-well plates) cells were transfected with siRNA specific for FPR2 (siFPR2, Thermo Fisher Scientific, Waltham, MA, USA). Negative control siRNA (siCONT) consisted of a pool of enzyme-generated siRNA oligonucleotides of 15–19 base pairs that were not specific for any known human gene (AllStars Neg. siRNA AF 488, Qiagen, Hilden, Germany) and showed no sequence similarity to FPR2. Briefly, siFPR2 or siCONT were used at a ratio of 1:6 and incubated for 15 min at room temperature. The siRNA complexed with the Hi-Perfect transfection reagent (Qiagen, Hilden, Germany) was then added drop-wise to a final concentration of 80-µM siRNA and incubated at 37 °C for 24–48 h hours. Trypan blue exclusion assay was performed to verify the viability of cells following siRNA transfection.