Figure 1.
Figure 1. Mature OCs express proapoptotic DRs. (A) CTSK and DR5 were immunostained in green and red, respectively, in a bone section. For staining OCs in bone, the tibiae of ICR-nu/nu mice (CLEA) were taken out, fixed for 2 days in 10% paraformaldehyde/PBS, and decalcified in 10% EDTA (pH 7.4) for 1 week. The tibiae specimens were embedded in paraffin, and then 5 μm thick serial sections were prepared. The deparaffinized sections were blocked with PBS containing 4.0% bovine serum albumin (PBS-BSA) for 1 hour at room temperature. To detect the localization of DR5 and CTSK in the tibia, the sections were incubated with anti-DR5 and anti-CTSK antibodies diluted 1:100 in PBS-BSA overnight at 4°C. After being washed 3 times with PBS, sections were reacted with Alexa Fluor 594–conjugated anti-rabbit IgG and Alexa Fluor 488–conjugated anti-mouse IgG secondary antibodies. The sections were observed under a fluorescence microscope (BX50, Olympus). Original magnification ×100. BM, bone marrow. (B) Murine RAW264.7 preosteoclastic cells were treated with sRANKL (50 ng/mL) for 72 hours, and DR5 mRNA expression was analyzed by RT-PCR (upper). Human monocytes from a healthy donor were treated with M-CSF (10 ng/mL) and sRANKL (50 ng/mL) for 5 days, and DR4 and DR5 mRNA expression was analyzed by RT-PCR (lower). GAPDH was used as an internal control. DR4 and DR5 mRNA expression was also determined using real-time PCR. GAPDH was used as an endogenous control to normalize each sample. Data were expressed as mean ± standard error. *P < .05. (C) Mature OCs derived from RAW264.7 cells were fixed and stained with anti-DR5 antibody (red, upper panels). F-actin and nuclei were stained with phalloidin (green) and Hoechst 33342 (Hoechst, red), respectively (lower panels). Samples were visualized with a fluorescence microscope (BX50, Olympus). Original magnification ×100. Bar represents 100 μm. (D) RAW264.7 cells (left) or mouse BMMs (right) were treated with sRANKL (50 ng/mL) for the indicated time periods, and then cell lysates were collected. The expression of DR5, c-FLIP, NFATc1, and CTSK was analyzed by western blotting. β-actin served as a loading control.

Mature OCs express proapoptotic DRs. (A) CTSK and DR5 were immunostained in green and red, respectively, in a bone section. For staining OCs in bone, the tibiae of ICR-nu/nu mice (CLEA) were taken out, fixed for 2 days in 10% paraformaldehyde/PBS, and decalcified in 10% EDTA (pH 7.4) for 1 week. The tibiae specimens were embedded in paraffin, and then 5 μm thick serial sections were prepared. The deparaffinized sections were blocked with PBS containing 4.0% bovine serum albumin (PBS-BSA) for 1 hour at room temperature. To detect the localization of DR5 and CTSK in the tibia, the sections were incubated with anti-DR5 and anti-CTSK antibodies diluted 1:100 in PBS-BSA overnight at 4°C. After being washed 3 times with PBS, sections were reacted with Alexa Fluor 594–conjugated anti-rabbit IgG and Alexa Fluor 488–conjugated anti-mouse IgG secondary antibodies. The sections were observed under a fluorescence microscope (BX50, Olympus). Original magnification ×100. BM, bone marrow. (B) Murine RAW264.7 preosteoclastic cells were treated with sRANKL (50 ng/mL) for 72 hours, and DR5 mRNA expression was analyzed by RT-PCR (upper). Human monocytes from a healthy donor were treated with M-CSF (10 ng/mL) and sRANKL (50 ng/mL) for 5 days, and DR4 and DR5 mRNA expression was analyzed by RT-PCR (lower). GAPDH was used as an internal control. DR4 and DR5 mRNA expression was also determined using real-time PCR. GAPDH was used as an endogenous control to normalize each sample. Data were expressed as mean ± standard error. *P < .05. (C) Mature OCs derived from RAW264.7 cells were fixed and stained with anti-DR5 antibody (red, upper panels). F-actin and nuclei were stained with phalloidin (green) and Hoechst 33342 (Hoechst, red), respectively (lower panels). Samples were visualized with a fluorescence microscope (BX50, Olympus). Original magnification ×100. Bar represents 100 μm. (D) RAW264.7 cells (left) or mouse BMMs (right) were treated with sRANKL (50 ng/mL) for the indicated time periods, and then cell lysates were collected. The expression of DR5, c-FLIP, NFATc1, and CTSK was analyzed by western blotting. β-actin served as a loading control.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal