Fig 2: Levels of OPG in third-generation osteoblasts following treatment with r-Mt cpn10 (10 µg/ml) for 2, 4, 8 and 24 h. (A) Levels of OPG mRNA following stimulation with control or r-Mt cpn10 (10 µg/ml) for 2, 4, 8 and 24 h. Total RNA was isolated from the harvested cells, transcribed to cDNA and quantified by reverse transcription-quantitative polymerase chain reaction analysis. The amount of OPG mRNA was compared with the amount of the reference gene (GAPDH) mRNA. Values are expressed as the mean ± intra-assay variation of duplicate analyses. (B) Western blotting of OPG protein expression following treatment with the control or r-Mt cpn10 (10 µg/ml) for 2, 4, 8 and 24 h. (C) Quantification of the level of OPG protein following treatment with the control or r-Mt cpn10 (10 µg/ml) for 2, 4, 8 and 24 h. Results are expressed as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001. r-Mt, recombinant Mycobacterium tuberculosis; cpn10, 10-kDa co-chaperonin; OPG, osteoprotegerin.

Fig 3: Effects of TF on the osteoblast phenotype.Mouse primary osteoblasts were isolated from the neonatal calvariae of 3- to 6-day-old WT mice, and osteogenic and osteoblast-related gene expression levels in the presence or absence of the indicated concentrations of TF with or without FVIIa (8 pg/mL) were quantified by qPCR. The mRNA levels of Runx2 (A), osterix (B), ALP (C), osteocalcin (D), type 1 collagen (E), RANKL (F), and OPG (G) and the RANKL/OPG ratio (H) were analyzed and normalized to GAPDH mRNA levels. ALP activity levels in cell homogenates were also quantified (I). Data represent the mean ± SEM of 6 experiments in each group. (J) Total protein was extracted from mouse osteoblasts treated with or without TF (50 pg/mL) and FVIIa (8 pg/mL). A Western blot analysis of osterix, RANKL, OPG, and ß-actin was then performed. Images represent experiments performed independently in triplicate. Protein signals were quantified by densitometry and adjusted by the density of ß-actin. Data represent the mean ± SEM of 3 experiments in each group.

Fig 4: Effects of LIRA on bone regeneration by regulating RANKL/OPG. (a) Immunohistochemical staining of RANKL and OPG in periodontal tissues. Scale bar, 100 µm. (b, c) Quantitative analysis of RANKL/OPG ratios. (d) The expression of Runx2 mRNA. (e) The expression of ALP mRNA. *P < 0.05; **P < 0.01.

Fig 5: RANKL/OPG ratio following treatment with r-Mt cpn10 (10 µg/ml) for 2, 4, 8 and 24 h. Results are expressed as the mean ± standard devation. *P<0.05 compared with the control. RANKL, receptor activator of nuclear factor-?B ligand; OPG, osteoprotegerin; r-Mt, recombinant Mycobacterium tuberculosis; cpn10, 10-kDa co-chaperonin.