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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Nature. 2010 Aug 4;466(7310):1110–1114. doi: 10.1038/nature09264

Figure 2. Rb regulates osteosarcoma-cell lineage plasticity in vitro and in vivo.

Figure 2

The differentiation potential of 3 different Osx-Cre;Rbfl/fl:p53fl/fl (DKO) and Osx-Cre:p53fl/fl (p53KO) OS cell lines was assessed 0, 7, 14, or 21 days after addition of differentiation media. a, Representative staining for: (left lane) alkaline phosphatase prior to differentiation induction; (middle lane) Alizarin Red to detect bone mineralization 14 days after culture in osteogenic-induction media and (right lane) Oil-Red-O to detect lipid droplets 14 days after culture in adipogenic-induction media. Expression of bone (Runx2, Alp, Coll1a and Bsp) and fat (Ap2, Pparγ, C/ebpα and Pgc1α) markers was assessed by qPCR of un-induced DKO (orange) and p53KO (black) OS cells. Bars represent the mean of three independent experiments (+/− SD). NS = not significantly expressed. b, Rb or control (Luc) shRNAs were expressed in the p53KO cell lines. Rb knockdown was confirmed by immunoprecipitation and qPCR showed that this caused downregulation of bone markers Bsp (and also Coll1a and Alp, data not shown), and upregulation of fat markers Pparγ (and also Ap2 and C/ebpα, data not shown) without culture in differentiating media. Bars represent the mean of three independent experiments (+/− SD). c, The osteogenic and adipogenic potential of shLuc- and shRb-p53KO cell lines was assessed 0, 7, 14 and 21 days after differentiation induction by Alizarin Red and Oil-Red-O staining. A representative timepoint (14 days) is shown. d, H+E staining of representative tumors derived from shLuc- and shRb-p53KO cell lines injected subcutaneously into immunocompromised mice. shRb-p53KO OS cells consistently (10/10 injections) yielded tumors that arose faster, and were more aggressive, than those arising from the parental p53KO OS controls (10 injections). Moreover, the shRb-p53KO OS derived tumors were frequently (6/10 injections) mixed lineage (top inset shows fat neoplasm; bottom inset bone/undifferentiated sarcoma), while the control shLuc-p53KO tumors were uniformly (10/10 injections) osteosarcomas. Additional analysis of these tumors (H+E, sirius red staining and Runx2 IHC) is shown in Supplementary Figure 4.