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Additional file 3: Figure S3. Immunoblotting analysis of the effect of OPG treatment on RANK expression. (A) An equal amount of membrane lysate proteins were used for immunoblotting analyses with antibodies to RANK (~90 kDa) and GAPDH (loading control; ~37 kDa). Protein levels were quantified by densitometry, corrected for the sample load based on GAPDH expression, and expressed as fold-decrease relative to the control lanes (RANKL and LPS). The results represent one of three experiments performed. (B) Uncropped raw data for the immunoblotting analyses shown in panel A are provided. Red rectangle indicates the proteins that are shown in panel A. The other lanes that were not marked by a rectangle in each autoradiogram represent different treatments which are not pertinent to the present studies.

Additional file 4: Figure S4. Effect of anti-TNF-α on the differentiation of osteoclasts. RAW cells subjected to differentiation in the presence of RANKL (A) and TNF-α (C) were treated with a TNF-α antibody for three days (B and D). Differentiation was blocked in cells treated with TNF-α antibody in TNF-α –treated cells (panel D) and not in RANKL treated cells (panel B). This result suggests that anti- TNF-α had no effect on the RANKL-RANK pathway involved in osteoclast differentiation. Representative phase-contrast microscopy images of TRAP-stained osteoclasts are shown. Cells were photographed under a 20X objective.

Additional file 1: Figure S1. Analysis of the potential osteoclastogenic effect of the LPS. (A) The diagrammatic sketch demonstrates the treatment strategy of RAW cells with RANKL and LPS. (B) And (C) representative images of TRAP stained osteoclasts (B) and RAW cells (C) in response to the treatment strategy shown in panel A. Images of different doses of LPS are shown in (C).

Additional file 7: Figure S7. Immunoblotting analysis of membrane levels of TNFR-1 (panel A) and TNFR-2 in response to RANKL (R) and LPS-treatment. Uncropped raw data for the immunoblotting analyses shown in Fig. 5A for the membrane (surface) levels of TNFR-1 (A) and TNFR-2 (B) are provided. White rectangle in A indicates the TNFR1 protein band (~55kDa) and red rectangle in B panel 1) indicate the TNFR-2 (~68kDa) protein band of interest. Four blots for TNFR-1 (A) and three blots for TNFR-2 (B) are shown. TNFR-1 and TNFR-2 bands were scanned in Un-Scan-IT software and provided as percent surface levels in (Fig. 5C and D) in the manuscript. (C) Immunoblotting analysis of membrane levels of TNFR-1 and TNFR-2 in cells treated with LPS and LPS/anti-TNF-α. Uncropped raw data (Two autoradiogram for each experiment) of the immunoblotting analyses shown in Fig. 5B are provided. Blots were scanned and fold change in the levels of TNFR-1 and TNFR-2 are provided in (Fig. 5C-H) in the manuscript. The red rectangle in panel 3 (top) indicate the TNFR-2 (~68kDa) protein band. TNFR1 and TNFR-2 bands were scanned and provided as fold change in the surface levels in (Fig. 5 E-H) in the manuscript.

Additional file 2: Figure S2. Analysis of the effect of OPG treatment on RANKL-stimulated osteoclastogenesis. (A) Identification of the time-dependent effect of OPG on RANKL-induced osteoclast differentiation. The diagrammatic sketch demonstrates the treatment strategy of RAW cells with RANKL and OPG (120 ng/ml). (B) Representative images of TRAP stained osteoclasts in response to the treatment strategy shown in panel A. (C) The number of TRAP +ve multinucleated osteoclasts were counted in all treatment groups. Statistical analysis was performed to compare the number in the late and early+late treatment groups to the control group (RANKL). One-way ANOVA was applied, and the values were expressed as mean ± SD. *P < 0.05 vs. the control group (C).

Additional file 5: Figure S5. Analysis of the RANK expression in LPS- and RANKL - mediated osteoclastogenesis. Uncropped raw data for the immunoblotting analyses shown in Fig. 2a are provided.