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Next, we generated the androgen responsive reporter plasmid containing PSA promoter and luciferase (PSA Lux). When LNCaP was transfected with PSA Lux and cultured under androgen deprived condition, increased luciferase activity of 2.5 fold was observed in the presence of bone marrow (HS 5) but not normal prostate stromal cell line (WPMY 1) (Figure 1A). When the second androgen responsive human CaP cell line 22Rv1 was used, a more modest but statistically significant increase in cell number was observed again in the presence of HS 5 bone stromal cells (Supplementary Figure 3A).
Bone morphogenetic protein type I receptors were knocked down using siRNA. Knockdown of ALK2 blocked BMP 6 mediated proliferation of LNCaP under androgen deprivation. Control experiments demonstrating the specificity of knockdown are shown in Supplementary Figure 3.
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