Deletion of TNF receptors potentiates RANKL-induced NO production and induction of iNOS expression. (A) Effect of RANKL on the production of NO in wild-type and TNF receptor-deleted macrophages. 1.0 × 10⁶ cells were treated with the indicated concentrations of RANKL for 12 hours. Culture medium was collected, and NO production was determined by using Griess reagent as described in “Materials and methods.” Numbers on top of the bars indicate percentage of change ± SD. (B) Effect of RANKL on the expression of iNOS in wild-type and TNF receptor-deleted macrophages. One million cells were treated with various concentrations of RANKL for 12 hours. Whole-cell extract (50 μg) was resolved by 7.5% SDS-PAGE and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-iNOS antibody as described in “Materials and methods.” The same membrane was reblotted with anti-β-actin antibody. (C) Effect of RANKL on the expression of COX-2 in wild-type and TNF receptor-deleted macrophages. 1.0 × 10⁶ cells were treated with the indicated concentrations of RANKL for 12 hours. Whole-cell extract (50 μg) was resolved by 7.5% SDS-PAGE and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-COX-2 antibody as described in “Materials and methods.” The same membrane was reblotted with anti-β-actin antibody. (D) Effect of RANKL on the cell viability of wild-type and TNF receptor-deleted macrophages. 5.0 × 10³ were seeded in 0.1 mL culture media in 96-well plates and exposed to the indicated concentrations of RANKL for 72 hours in triplicate, and then cell viability was determined using the MTT assay as described in “Materials and methods.” Numbers indicate percentage of change ± SD. (E) RANKL induces caspase-activated PARP cleavage in TNF receptor-deleted macrophages. 1.0 × 10⁶ cells were treated with the indicated concentrations of RANKL for 24 hours. Whole-cell extract (50 μg) was resolved by 7.5% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and probed for PARP using anti-PARP antibody as described in “Materials and methods.” (F) Antiproliferative effects of RANKL require NO production. 1.0 × 10⁴ p60^-/-p80^-/- macrophages were seeded in 0.1 mL culture media in 96-well plates, preincubated with 100 μM L-NAME for 1 hour, and then exposed to 1 nM RANKL in triplicate. After 72 hours, cell viability was determined by the MTT method as described in “Materials and methods.” Numbers on top of the bars indicate percentage of change ± SD.