Characterization of the bone and osteoblast pathology. A. Photograph of the bone biopsy. B. Steady state SMS mRNA levels relative to GAPDH expression in cultured fibroblasts and osteoblasts. The patient’s cells did not differ significantly from controls. Data were derived by qRT-PCR analysis of 3 independent extractions of total RNA. C. Immunoblot showing steady state SMS protein expression in patient and control osteoblasts. ß-tubulin is shown as a loading control. D. Graph showing steady state SMS protein levels in the patient and control hBMSCs relative to ß-tubulin levels; there was no significant difference. The data are based on 3 independent experiments for each cell line. E-J. Immunofluorescent detection of SMS protein subcellular distribution in unaffected (E-G) and Patient II-1 (H-J) hBMSCs. SMS protein is shown in red and the nucleus is shown in blue. K. Graph quantifying immunoblot detected steady state SMS protein levels in the cytoplasm and nuclei of patient and control hBMSCs. The cytoplasmic expression was normalized to β-tubulin expression and the nuclear expression to p84 expression. L. Polyamine quantification in fibroblasts and osteoblasts. Note that the patient hBMSCs have a more striking imbalance of spermidine and spermine levels than do the patient fibroblasts, * p < 0.05, *** p < 0.005. M. Osteogenic potential of bone marrow stromal cells (hBMSCs) isolated from Patient II-1 sample is markedly lower than that of an unaffected control (cnt). The hBMSCs were seeded in triplicates (6x104/12-well) and either kept untreated (-) or treated (+) with osteogenic differentiation media (see Methods) for 18 days. After the treatment, cells were fixed and were stained with Alizarin Red S to check for calcium deposition, a marker of osteogenic differentiation.