Gr sirna

For in vitro siRNA transfection, GR siRNA (Santa Cruz Biotechnology; sc-35506) and Control siRNA (Santa Cruz Biotechnology; sc-37007) were transfected into POCs according to the manufacturer’s guidelines. Briefly, RAW264.7 cells were induced to differentiate into POCs by culture for 3 days in the presence of RANKL and M-CSF. POCs were transfected with either GR siRNA or Control siRNA duplex diluted in siRNA transfection medium (Santa Cruz Biotechnology; sc-36868) and mixed with siRNA transfection reagent (Santa Cruz Biotechnology; sc-29528) into siRNA transfection medium after incubation for 30 min at room temperature. Cells were incubated for 6 hours at 37°C in a 5% CO₂ incubator. Transfection mixture was removed and replaced with Dulbecco’s Modified Eagle Medium (DMEM) culture medium containing 10% FBS, RANKL 60 ng/mL, and M-CSF 30 ng/mL, and cells were incubated for an additional 24 hours. Conditioned medium, chromatin, cell protein, and RNA were collected for ELISA, chromatin immunoprecipitation (ChIP), Western blot analysis, and RT-PCR.