Neon transfection method

Gene knockdowns were performed with Silencer Select siRNA oligos (Thermo Fisher Scientific) specific for PTGER2 (s11449) or PTGER4 (s60395), or combinations thereof, using the NEON transfection method (Thermo Fisher Scientific) as previously described^{55 (link)}. Briefly, hBMSCs (1 × 10⁵ cells) were transfected with up to 20 nM of siRNAs or negative control siRNA (Negative Control-1), and seeded in cell culture plates with fresh growth medium (without antibiotics) for 24 h at 37 °C, 5% CO₂. Medium was then replaced with osteogenic induction medium, and knockdown efficiency confirmed at selected time points by RT-qPCR.

U87MG cells (ATCC, Manassas, VA, USA) were cultured in DMEM (Welgene, Gyeongsan, South Korea, and PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (Gibco-BRL, Grand Island, NY, USA and PAN Biotech) and 1% penicillin–streptomycin (HyClone, Logan, UT, USA, and PAN Biotech). Cells were grown at 37 °C in a humidified atmosphere of 5% CO₂. Mycoplasma testing was conducted periodically. Cells were subjected to transient transfection with expression plasmids using the Effectene Reagent (QIAGEN, Hilden, Germany) or the Neon transfection method (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturers’ manual. The following reagents were used at doses indicated in the figure legends: Rapamycin were obtained from Invivogen, Hong Kong; recombinant rat PDGF–BB (520-BB-050) was purchased from R&D Systems, Minneapolis, MN, USA; chlorpromazine was purchased from Sigma-Aldrich, St. Louis, MO, USA; Pitstop 2 was purchased from Abcam, Cambridge, United Kingdom; and CellMask Plasma Membrane Stain was purchased from Life Technologies, Carlsbad, CA, USA.