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Figure 3. PKC pathway is partially involved in PTHrP-induced TRAP⁺ cell formation in co-cultures. (A) Co-cultures of mouse spleen cells and human PDL cells were pre-treated with or without PD98059 (10 µM) (ERK inhibitor), NS-398 (10 µM) (COX2 inhibitor), SB203580 (10 µM) (p38 MAPK inhibitor), and Ro-32-0432 (1 µM) (PKC inhibitors) for 30 min, followed by incubation with PTHrP (10^–8 M) together with Dex for 10 days. TRAP⁺ cells were counted and expressed as number of TRAP⁺ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. PTHrP-treated cultures. (B) Human PDL cells were pre-treated with or without SB203580 (10^–7, 10^–6 M) for 30 min, followed by incubation with PTHrP (10^–8 M) for 72 hrs. Total RNA was isolated from PDL cells, and expression levels of RANKL, OPG, and GAPDH mRNA were measured by RT-PCR analysis. Numbers below the gels represent n-fold increase in intensity of RANKL or OPG relative to corresponding GAPDH mRNA signals. Similar results were obtained in 3 independent experiments. (C) We cultured mouse bone marrow cells (BMCs) with M-CSF for 3 days to prepare bone marrow macrophages (BMMs). BMMs were pre-treated with or without SB03580 (10^–7, 10^–6 M) for 30 min, followed by incubation with RANKL (100 ng/mL) in the presence of M-CSF (100 ng/mL) for 3 days. Data shown are number of TRAP⁺ MNCs per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. RANKL-treated cultures. (D) Co-cultures of mouse spleen cells and human PDL cells were pre-treated with or without Ro-32-0432 (0.1 and 1 µM) for 30 min, followed by incubation with PTHrP (10^–8 M) together with Dex for 10 days. TRAP⁺ cells were counted and expressed as number of TRAP⁺ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. PTHrP-treated cultures. (E) Human PDL cells were pre-treated with or without Ro-32-0432 (0.1, 1 µM) for 30 min, followed by incubation with PTHrP (10^–8 M) for 72 hrs. Total RNA was isolated from PDL cells, and expression levels of RANKL, OPG, and GAPDH mRNA were measured by RT-PCR analysis. Numbers below the gels represent n-fold increase in intensity of RANKL or OPG relative to corresponding GAPDH mRNA signals. Similar results were obtained in 3 independent experiments. (F) We cultured mouse BMCs with M-CSF for 3 days to induce BMMs. BMMs were pre-treated with or without Ro-32-0432 (0.1, 1 µM) for 30 min, followed by incubation with RANKL (100 ng/mL) in the presence of M-CSF (100 ng/mL) for 3 days. Mouse BMCs were pre-treated with or without Ro-32-0432 (0.1, 1 µM) for 30 min, followed by incubation with RANKL (100 ng/mL) in the presence of M-CSF (100 ng/mL) for 5 days. Data shown are number of TRAP⁺ MNCs per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. RANKL-treated cultures. (G) Mouse BMCs were pre-treated with or without Ro-32-0432 (0.1, 1 µM) for 30 min, followed by incubation with M-CSF (100 ng/mL) for 3 days. After 3 days, cells were stained with FITC-conjugated anti-c-fms antibody, and c-fms⁺ cells were counted by flow cytometry (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. M-CSF-treated cultures.

J DENT RES, Vol. 84, No. 4, 329-334 (2005)
DOI: 10.1177/154405910508400407

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