Image pro plus version 4

BMCs (1 × 10⁷ cells) and primary osteoblasts (1 × 10⁶ cells) were seeded on collagen gel-coated culture dishes and cultured for 7 days in the presence of 10^− 8 M 1,25-dihydroxyvitamin D₃ (Sigma) and 10^− 6 M prostaglandin E₂ (PGE₂) (Sigma). The co-cultured cells were detached by 0.1% collagenase treatment at 37 °C for 10 min and were then replated on hydroxyapatite-coated plates (Corning, Corning, NY, USA) for 24 h and dentin slices for 48 h. Cells attached to the plates or dentin slices were removed and the total resorption pits were photographed by microscopy and analyzed using Image-Pro Plus version 4.0 (Media Cybernetics, Silver Spring, MD, USA).

Mature OCs were prepared from the co-culture of BMCs and primary OBs as described previously^{16 (link)}. Briefly, BMCs (1 × 10⁷ cells) and primary OBs (1 × 10⁶ cells) were cultured in collagen gel-coated culture dishes in the presence of 10⁻⁸ M VitD₃ and 10⁻⁶ M PGE₂ for 9 days. Co-cultured mature OCs were detached using 0.1% collagenase at 37 °C for 10 min and re-seeded in 48-well plates, hydroxyapatite-coated plates (Corning, NY, USA), and dentin slices. After 1 h, the cells were transfected with the indicated retrovirus or small interfering RNA (siRNA) as described above and further cultured in the presence of RANKL (100 ng/mL). After 48 h, the cells re-seeded in 48-well plates were stained with TRAP solution to detect the survival of OCs. The cells re-seeded in hydroxyapatite-coated plates and dentin slices were completely removed using 10% sodium hypochlorite after 24 and 48 h, respectively. Additionally, dentins were stained with hematoxylin to detect resorption pits. The total number of resorption pits was determined under a microscope and were quantified using Image-Pro Plus version 4.0 (Media Cybernetics, Silver Spring, MD, USA).

Experimental mice were sacrificed on day 15 and long bones were obtained. To generate osteoclasts or osteoblasts, 5×10⁴ cells were plated per well in 24-well tissue culture plates and treated with different doses of M-CSF (5 mg/ml) or RANKL (5 mg/ml), respectively as described previously (24 (link)) Cells cultured in the presence of 10⁻⁶M 1,25-dihydroxyvitamin D₃ and 10⁻⁶ M prostaglandin E₂ (Sigma-Aldrich; Merck, Darmstadt, Germany) for 7 days at 37°C. The total bone resorption was measured using ImagePro Plus version 4.0 (Media Cybernetics, Inc., Silver Spring, MD, USA) as described previously (25 (link)).