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. 2013 Sep;183(3):796–807. doi: 10.1016/j.ajpath.2013.06.008

Figure 3.

Figure 3

Omniscan-induced cells have an osteogenic phenotype. A: Osteoblast-lineage marker expression by PBMCs treated with 0.5 mmol/L Omniscan using immunofluorescence. Omniscan-induced, human PBMC-derived adherent cells express the macrophage markers CD163 (red) and CD206 (green) as shown by immunofluorescence. Omniscan-induced cells significantly expressed osteopontin (red), osteocalcin (red), procollagen-1 (green), and were double-positive for osteocalcin and procollagen-1 (yellow) compared with control cells. The majority of Omniscan-induced cells expressed FGF23 (green). Scale bars: 100 μm. B: Protein expression by Omniscan-induced cells. Western blot analysis of human PBMCs treated (T) with 0.5 mmol/L Omniscan or untreated (C) were performed using total cell lysates from adherent cells, as described in Materials and Methods. Detection for CD163, CD206, H-ferritin, ferroportin, procollagen-1, osteocalcin (OCN), osteopontin, and FGF23 expression was performed by immunoblotting. The representative blots in the figure show increased expression of these markers by Omniscan-induced cells compared with controls. The same blots were stripped and reprobed with β-actin antibody. C: Graphic representation of densitometric scans of Western blots. Data presented were derived from six separate experiments (donors) with or without 0.5 mmol/L Omniscan treatment, and means ± SEM of band intensities in fold induction above control of representative proteins were plotted. On the plot, 1 on the x axis represents the controls. ∗P < 0.05, ∗∗P < 0.01 compared with controls. D: Quantitative gene expression by Omniscan-induced cells. cDNA was synthesized from RNA isolated from Omniscan-induced adherent cells. Solaris quantitative real-time PCR gene expression reagents (Dharmacon) were used to detect CD163, procollagen-1, ferroportin, hepcidin, Runt-related transcription factor 2 (Runx2), Osterix (OSX, SP7), and reference genes. cDNA and relative gene expression was calculated from quantification cycle (Cq) values using a delta deltaCq. The figure shows increased gene expression of CD163, ferroportin, procollagen-1, osteocalcin, and osteoblast transcription factors (Runx-2 and OSX) by Omniscan-induced cells compared with controls. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001, for Omniscan-treated cells compared with controls.