CID induced osteoclastogenesis in RAW264.7+iRANK cells is OPG-independent.

<p>TRAP-positive multinucleated cells (MuNC) after treatment of RAW264.7+iRANK cells with 10 nM AP20187 (A) or RAW264.7 cells with 1 nM RANKL (B) in the presence of increasing concentrations of OPG. TRAP-positive multinucleated cells (MuNC) were counted over 4 high power fields of view and averaged over 3 wells. *p<0.05 compared to 0 nM OPG.</p

NF-κB signaling.

<p>NF-κB dependent signaling in engineered osteoclasts. RAW264.7 and RAW264.7+iRANK cells were transiently transfected with a luciferase reporter construct containing NF-κB sites derived from Igκ promoter driving the luciferase gene and a Renilla luciferase construct as the internal control. NF-κB activation was measured in RAW264.7+iRANK cells stimulated with AP20187 (A), and RAW264.7 cells stimulated with RANKL (B) or LPS (C) for 2 and 4 h. Data are average relative light units (RLU) per µg protein +/− SD. *p<0.05.</p

Schematic representation of CID-inducible cytoplasmic RANK (iRANK) lentiviral construct and Western blot detecting construct.

<p>A) LTR = long terminal repeat; RRE = rev response element; cPPT = central polypurine tract; EF1a = elongation factor 1-Alpha; EGFP = green fluorescent protein; I = IRES; M = myristoylation; F36V = FKBP12; F36V' = modified FKBP12; cRANK = cytoplasmic domain of RANK; WPRE = WHP posttranscriptional regulatory element. B) Western blot probed with an antibody for FKBP12 showing overexpression of the iRANK construct in RAW264.7+iRANK cells migrating around 70 kDa. Cells transduced just with the F36V' domains (RAW264.7+F2) show a band around 30 kDa. No bands appear in the untransduced RAW264.7 cells.</p

Cell survival study.

<p>RAW264.7 cells and RAW264.7+iRANK cells were treated with either RANKL (40 ng/ml) or AP20187 (50 nM) for 4 days to allow osteoclasts to form. The supplemented media was removed, and cells were cultured for additional 0, 3 or 5 days in the presence (A) or absence (B) of inducers, and the number of TRAP-positive multinucleated cells (MuNC) per well was counted and averaged over 4 wells. *p<0.05.</p

CID induced osteoclasts resorbed a two-dimensional mineralized substrate.

<p>(A) RAW264.7+iRANK cells were treated with either RANKL (100 ng/ml) or AP20187 (100 nM) on Osteologic discs. After 10 days, resorption lacunae were visualized by von Kossa staining. (B) The percent resorbed area per disc was measured and analyzed using ImageJ. Three experiments were averaged. Y-axis shows the % resorption per disc. *p<0.05.</p

CID-responsiveness of the iRANK construct.

<p>RAW264.7+iRANK cells were cultured in medium containing vehicle (EtOH), RANKL, or 0.1–50 nM AP20187 for 4 days and the cells were stained for TRAP. RANKL and AP20187 induced multinucleated TRAP-positive cells were observed (purple staining) (scale bars = 100 µm).</p

CID induced osteoclasts resorbed a three-dimensional mineralized substrate.

<p>(A) Mineralized fibrin scaffolds were seeded with control RAW264.7 cells (white bars) or iRANK transduced RAW264.7 cells (black bars), or no cells (hatched bar). The scaffolds were weighed at various time points (days 2, 5, 8 and 11). The scaffolds without cells were incubated in media for 11 days. Mass loss was calculated by subtracting the final mass from the initial mass. *p <0.05. (B) RAW264.7+iRANK were cultured with AP20187 in the fibrin scaffolds for 8 days. H&E staining (left panel) and adjacent TRAP staining (right panel) indicate the differentiation of osteoclasts within the scaffolds (arrows) (scale bars = 50 µm).</p

CID induced osteoclasts formed on native mineralized substrates.

<p>(A) TRAP-positive osteoclasts on human dentin slices. Dentin slices created from human teeth were seeded with RAW264.7+iRANK cells and cultured in the presence of AP20187 for 9 days. The slides were fixed and stained for TRAP and multinucleated TRAP-positive cells were shown in purple (arrow) (scale bar = 100 µm). (B) Confocal micrograph of multinucleated osteoclasts on mouse calvarial disc. Calvarial discs were seeded with RAW264.7+iRANK cells and osteoclast formation was induced by the addition of AP20187. Cells were imaged by confocal microscope; blue fluorescence indicates nuclei by Hoechst stain, while green fluorescence indicates GFP expression in RAW264.7+iRANK cells (scale bar = 50 µm).</p