RANKL knockdown mediated by antisense RNA in PLB-985 neutrophil-like cells inhibits osteoclastogenesis. Flow cytometric analysis of RANKL expression by dbcAMP-differentiated PLB-985 cells transfected (A) with control nonsilencing antisense RNA (Ctl siRNA), or (B) with RANKL antisense RNA (RANKL siRNA). RANKL expression was examined 48 hours after transfection by incubating cells with an anti–human RANKL mAb followed by an FITC-conjugated anti–mouse F(ab′)₂ antibody. Isotype controls are dotted tracings. (C) Histograms represent means (± SEM) of percentage of cells expressing RANKL (n = 3 independent experiments). (D) PLB-985 neutrophil-like cells (10⁶/well) transfected with Ctl antisense RNA or RANKL antisense RNA for 48 hours were fixed with 2% PFA, repeatedly washed, and added to cultures of human peripheral blood monocytes in 96-well plates. After 10 days, TRAP-positive multinucleated cells were counted. Results are expressed as means (± SEM) of the number of multinucleated TRAP-positive cells/well (n = 3 independent duplicated experiments). (Inset) TRAP-positive multinucleated cell obtained after cocultures of human peripheral blood monocytes with transfected dbcAMP-differentiated PLB-985 cells (magnification, ×200). (E) PFA-fixed PLB-985 neutrophil-like cells transfected with Ctl antisense RNA or RANKL antisense RNA, were added to mature OCs on Osteologic discs. After 10 days of cocultures, cells were removed and surfaces of resorption (mm²) were measured by light microscopy using Imagepro software. Results are expressed as means (± SEM) of resorption area (n = 3 independent duplicated experiments). Statistical analysis: paired t test, *P < .05 (Ctl antisense RNA vs RANKL antisense RNA).