Figure 3. Silencing HOTAIR expression significantly diminishes BMP9-induced osteogenic differentiation of iMAD cells in vitro. (A) Silencing of HOTAIR impairs the endogenous and BMP9-induced activity of ALP in iMADs. Subconfluent iMAD-Ctrl and iMAD-KD cells were infected with Ad-GFP or Ad-BMP9. Qualitative histochemical staining (a) and quantitative bioluminescence assay (b) were done at days 3, 5, and 7 after infection. (B) HOTAIR knockdown significantly diminishes BMP9-induced mineral deposition of iMADs. Subconfluent iMAD-Ctrl and iMAD-KD cells were infected with Ad-GFP or Ad-BMP9 and cultured in mineralization medium for 12 days, and stained with alizarin red S (a). Alizarin red S quantification (b) was done using the ImageJ program. (C) Silencing of HOTAIR down-regulates the endogenous and BMP9-induced expression of osteogenic differentiation markers (a), and also diminishes BMP9-induced chondrogenic and adipogenic differentiation markers in iMADs (b). Subconfluent iMAD-Ctrl and iMAD-KD cells were infected with Ad-GFP or Ad-BMP9. Total RNA was isolated at 48 h and subjected to TqPCR analysis using gene-specific primers for mouse Runx2, Ocn, Osx, Sox9 and Ppar-γ. All assays were done in triplicate. Representative images are shown. “*” p < 0.05, iMAD-Ctrl+GFP group vs iMAD-KD+GFP group. “**” p < 0.05, iMAD-Ctrl+BMP9 group vs iMAD-KD+BMP9 group.