Figure 3.
Figure 3. c-FLIP knockdown impairs osteoclastogenesis and resumes TRAIL-induced apoptosis in mature OCs. (A) RAW264.7 cells were pretreated with sRANKL (50 ng/mL) for 48 hours, mouse BMMs were pretreated with M-CSF (20 ng/mL) and sRANKL (50 ng/mL) for 48 hours, and human PBMCs were pretreated in quadruplicate with M-CSF (10 ng/mL) and sRANKL (50 ng/mL) for 4 days. After the pretreatment, the media were changed, and TRAIL was added at the indicated concentrations. RAW264.7 cells, mouse BMMs, and human PBMCs were further cultured for 4, 7, and 14 days, respectively. The numbers of TRAP-positive MNCs were counted. Data were expressed as mean ± SD. *P < .05. The representative photos of TRAP staining are shown. Bars represent 100 μm. Original magnification ×100. Bone resorption activity was also analyzed using Osteo assay plates. Resorption area was visualized by von Kossa staining, and observed using a light microscope (BX50, Olympus). The results were expressed as % area of bone resorption. The cell lysates were collected, and NFATc1 and CTSK were analyzed by western blotting. β-actin served as a loading control. (B) c-FLIP siRNA or control siRNA was transfected into RAW264.7 cells. After the transfection, the cells were treated with sRANKL (50 ng/mL) for 4 days. The cells were then washed and treated with the indicated concentrations of TRAIL for 48 hours. The protein levels of c-FLIP were analyzed by western blotting (left). β-actin was used as a protein loading control. The numbers of TRAP-positive MNCs were counted. To identify apoptotic cells, the cells were stained with TUNEL staining. The TUNEL-positive multinuclear cells were counted. Data were expressed as mean ± SD. *P < .05. (C) Mature OCs from RAW264.7 cells and MM.1S cells were starved with 1% FBS for 24 hours and then incubated with TRAIL at 100 ng/mL for the indicated time periods. The cell lysates were collected and an equal amount of each protein lysate was incubated with anti-FADD antibody. Complex II formation was analyzed by western blotting with anti-RIP1, anti-TRAF2, and anti-FADD antibodies. (D) RAW264.7 cells transfected either c-FLIP or control siRNA were treated with sRANKL (50 ng/mL) for 48 hours. The cells were then washed and treated with at 100 ng/mL for the indicated time periods. The complex II formation was analyzed.

c-FLIP knockdown impairs osteoclastogenesis and resumes TRAIL-induced apoptosis in mature OCs. (A) RAW264.7 cells were pretreated with sRANKL (50 ng/mL) for 48 hours, mouse BMMs were pretreated with M-CSF (20 ng/mL) and sRANKL (50 ng/mL) for 48 hours, and human PBMCs were pretreated in quadruplicate with M-CSF (10 ng/mL) and sRANKL (50 ng/mL) for 4 days. After the pretreatment, the media were changed, and TRAIL was added at the indicated concentrations. RAW264.7 cells, mouse BMMs, and human PBMCs were further cultured for 4, 7, and 14 days, respectively. The numbers of TRAP-positive MNCs were counted. Data were expressed as mean ± SD. *P < .05. The representative photos of TRAP staining are shown. Bars represent 100 μm. Original magnification ×100. Bone resorption activity was also analyzed using Osteo assay plates. Resorption area was visualized by von Kossa staining, and observed using a light microscope (BX50, Olympus). The results were expressed as % area of bone resorption. The cell lysates were collected, and NFATc1 and CTSK were analyzed by western blotting. β-actin served as a loading control. (B) c-FLIP siRNA or control siRNA was transfected into RAW264.7 cells. After the transfection, the cells were treated with sRANKL (50 ng/mL) for 4 days. The cells were then washed and treated with the indicated concentrations of TRAIL for 48 hours. The protein levels of c-FLIP were analyzed by western blotting (left). β-actin was used as a protein loading control. The numbers of TRAP-positive MNCs were counted. To identify apoptotic cells, the cells were stained with TUNEL staining. The TUNEL-positive multinuclear cells were counted. Data were expressed as mean ± SD. *P < .05. (C) Mature OCs from RAW264.7 cells and MM.1S cells were starved with 1% FBS for 24 hours and then incubated with TRAIL at 100 ng/mL for the indicated time periods. The cell lysates were collected and an equal amount of each protein lysate was incubated with anti-FADD antibody. Complex II formation was analyzed by western blotting with anti-RIP1, anti-TRAF2, and anti-FADD antibodies. (D) RAW264.7 cells transfected either c-FLIP or control siRNA were treated with sRANKL (50 ng/mL) for 48 hours. The cells were then washed and treated with at 100 ng/mL for the indicated time periods. The complex II formation was analyzed.

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